Ctenopharyngodon Idella Kidney Cells Research Articles

MicroRNA-30e-3p regulates the inflammatory response by targeting the gimap8 gene in Ctenopharyngodon idella kidney (CIK) cells

Recent studies have increasingly linked miRNAs with the modulation of inflammatory responses and immunosuppressive activities. This investigation reveals that mir-30e-3p selectively binds to and modulates gimap8, as demonstrated by luciferase reporter assays and qPCR analyses. Upon LPS stimulation of CIK cells, mir-30e-3p expression was notably elevated, inversely correlating with a decrease in gimap8 mRNA levels. Overexpression of mir-30e-3p attenuated the mRNA levels of pro-inflammatory cytokines beyond the effect of LPS alone, suggesting a regulatory role of mir-30e-3p in inflammation mediated by the gimap8 gene. These insights contribute to our understanding of the complex mechanisms governing inflammatory and immune responses.

Integrative transcriptome analysis reveals alternative polyadenylation potentially contributes to GCRV early infection.

Grass carp reovirus (GCRV), a member of the Aquareovirus genus in the Reoviridae family, is considered to be the most pathogenic aquareovirus. Productive viral infection requires extensive interactions between viruses and host cells. However, the molecular mechanisms underlying GCRV early infection remains elusive. In this study we performed transcriptome and DNA methylome analyses with Ctenopharyngodon idellus kidney (CIK) cells infected with GCRV at 0, 4, and 8 h post infection (hpi), respectively. We found that at early infection stage the differentially expressed genes related to defense response and immune response in CIK cells are activated. Although DNA methylation pattern of CIK cells 8 hpi is similar to mock-infected cells, we identified a considerable number of genes that selectively utilize alternative polyadenylation sites. Particularly, we found that biological processes of cytoskeleton organization and regulation of microtubule polymerization are statistically enriched in the genes with altered 3'UTRs. Our results suggest that alternative polyadenylation potentially contributes to GCRV early infection.

Comparative Transcriptomic Analysis Revealed Potential Differential Mechanisms of Grass Carp Reovirus Pathogenicity.

Grass carp reovirus (GCRV), one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella), can lead to grass carp hemorrhagic disease (GCHD). Currently, GCRV can be divided into three genotypes, but the comparison of their pathogenic mechanisms and the host responses remain unclear. In this study, we utilized the Ctenopharyngodon idella kidney (CIK) model infected with GCRV to conduct comparative studies on the three genotypes. We observed a cytopathic effect (CPE) in the GCRV-I and GCRV-III groups, whereas the GCRV-II group did not show any CPE. Moreover, a consistent trend in the mRNA expression levels of antiviral-related genes across all experimental groups of CIK cells was detected via qPCR and further explored through RNA-seq analysis. Importantly, GO/KEGG enrichment analysis showed that GCRV-I, -II, and -III could all activate the immune response in CIK cells, but GCRV-II induced more intense immune responses. Intriguingly, transcriptomic analysis revealed a widespread down-regulation of metabolism processes such as steroid biosynthesis, butanoate metabolism, and N-Glycan biosynthesis in infected CIK cells. Overall, our results reveal the CIK cells showed unique responses in immunity and metabolism in the three genotypes of GCRV infection. These results provide a theoretical basis for understanding the pathogenesis and prevention and control methods of GCRV.

MiR-130a targets CiGadd45bb to modulate the inflammatory response to bacterial infection in Ctenopharyngodon idella kidney (CIK) cells

Septicemia is a systemic inflammatory response to bacterial infection that results in a hyper-inflammatory state, which could lead to septic shock and death in grass carp (Ctenopharyngodon idella). The aim of this study was to determine the underlying mechanism of microRNA (miR-130a) in bacteria-infected grass carp. Expression levels of miR-130a against Aeromonas hydrophila (A. hydrophila) infection in Ctenopharyngodon idella kidney cells (CIK) were analyzed. Luciferase reporter assay, quantitative reverse transcription–polymerase chain reaction were performed to explore whether Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 (CiGadd45bb) was a target of miR-130a. MiR-130a mimic, inhibitor and miR-control were transfected to CIK respectively. After transfection, the expression levels of proinflammatory genes were determined. Here we show that CiGadd45bb as a target of miR-130a. We also confirmed that miR-130a levels were significantly higher after being stimulated for 4 h and lower after 12 h (P < 0.01). Overexpressing miR-130a strikingly inhibited p38, JNK, ERK and TNF-a genes (P < 0.01) and silencing miR-130a activated p38, JNK, ERK, TNF-a, IFN and IL-8 (P < 0.01). Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-130a in grass carp.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Eucalyptol relieves the toxicity of diisobutyl phthalate in Ctenopharyngodon idellus kidney cells through Keap1/Nrf2/HO-1 pathway: Apoptosis-autophagy crosstalk and immunoregulation

Diisobutyl phthalate (DiBP), one of the commonly used plasticizers in industry, is an endocrine disruptor and environmental contaminant that can persist in water and threaten the health of aquatic creatures. Eucalyptol (Euc), a monoterpenoid extracted from plants, has been proved to have anti-inflammatory, antioxidant, and detoxification properties. However, the protective mechanism of Euc against cell injury caused by DiBP exposure and the involvement of apoptosis, autophagy, and immunity remains unknown. In the current investigation, 27.8 μg/mL DiBP or/and 20 μM Euc has been applied to Ctenopharyngodon idellus kidney (CIK) cells for 24 h. The findings showed that exposure to DiBP raised intracellular ROS levels, inducing oxidative stress, and enhanced the rate of apoptosis as well as the expression of the apoptotic markers Bax, Caspase3, Caspase9, and Cytc while decreasing the expression of Bcl-2. Furthermore, DiBP inhibited IL-2, IFN-γ, Hepcidin-1, and β-defensin expression and elevated TNF-α, and IL-1β levels, causing immune dysfunction. DiBP and Euc co-treatment significantly activated the Keap1/Nrf2/HO-1 pathway, restored antioxidant enzyme activity, and elevated autophagy pathway-associated genes ATG5, Beclin1, and LC3B decreased p62 expression, enhanced cell autophagy, reduced apoptosis, and improved immunity. In conclusion, Euc promotes autophagy, alleviates DiBP-induced apoptosis, and improves immunological dysfunction in CIK cells by regulating the Keap1/Nrf2/HO-1 pathway. These results demonstrated the threat of DiBP exposure to fish while providing a theoretical foundation for using Euc in aquaculture.

First identification of Nocardia seriolae GapA adhesion function and its three B-cell epitopes with cell-binding activity.

Fish nocardiosis mainly caused by Nocardia seriolae (N. seriolae) is a serious threat to aquaculture. Bacterial adhesion to host cells mediated by adhesin is an initial step of pathogenesis. But it is not clear whether glyceraldehyde-3-phosphate dehydrogenase (GapA) is an adhesin of N. seriolae. Here, recombinant GapA protein (rGapA) was prokaryotic expressed, and its role in the bacterial adhesion to Ctenopharyngodon idella kidney cells was investigated by indirect immunofluorescence, protein-binding assay and adhesion inhibition assay. The results showed that an obvious green fluorescence was observed on the surface of the cells co-incubated with rGapA protein; the cytomembrane proteins of the cells pretreated with rGapA could react with anti-rGapA antibody; and the antibody significantly inhibited the adhesion ability of the bacteria. Subsequently, B-cell linear epitopes of GapA protein were identified by using a immunoinformatics approach combined with peptide ELISA and Western blot for the first time. It was found that four predicted epitopes (Ep58-69 , Ep139-150 , Ep186-197 , Ep318-329 ) could all react with anti-rGapA antibody and obviously inhibit the immunoreactivity between rGapA and anti-rGapA antibody, and they were confirmed as indeed B-cell linear epitopes of the protein. Furthermore, flow cytometry analysis found the percentage of positive cells co-incubated with FITC-labelled epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) was significantly higher than those in the FITC-labelled Ep58-69 , unrelated control peptide and cell control. Collectively, GapA is an adhesin of N. seriolae, and epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) possess cell-binding activity, which are potential candidates for developing a multiple epitopes-based adhesin vaccine against fish nocardiosis.

Fatty hepatocytes-derived exosomal miR-122 reduces immune function and antioxidant defence in Ctenopharyngodon idella kidney (CIK) cells

Exosomes are important for intercellular “cross talk”, but the role of exosomes in communication between hepatocytes and C. idella kidney (CIK) cells remains unknown. In this study, we detected the changes in factors related to immune and oxidative stress to investigate the molecular mechanism by which fatty hepatocyte-derived exosomes (OA-Exos) reduced immunity and induced oxidative stress in CIK cells. After incubation of CIK cells by OA-Exos for 24 h, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) and interleukin-1β (IL-1β) were significantly upregulated in the OA-Exos group (P < 0.05), and Mn superoxide dismutase (Mn-SOD) and heme oxygenase-1 (HO-1) were significantly downregulated (P < 0.05). Surprisingly, miR-122 expression was also significantly elevated after OA-Exos incubation. We further identified the expression of miR-122 and found that it was notably increased in OA-Exos compared to hepatocyte-derived exosomes (Exos). Then we transfected CIK cells with miR-122 mimic, consistently, the expression of inflammatory cytokines was also significantly elevated (P < 0.05), and the expression of glutathione peroxidase (GPx), HO-1, and Mn-SOD were dramatically decreased (P < 0.05). Furthermore, HO-1 was improved to be a direct target of miR-122, and transfection with HO-1 siRNA indicated that changes in inflammatory cytokines and genes related to oxidative stress were consistent with the above results of CIK cells incubated with OA-Exos and miR-122 mimic. We concluded that OA-Exos may, through the miR-122/HO-1 pathway, reduce immune function and antioxidant defence in CIK cells.

Melatonin administration alleviates 2,2,4,4-tetra-brominated diphenyl ether (PBDE-47)-induced necroptosis and secretion of inflammatory factors via miR-140-5p/TLR4/NF-κB axis in fish kidney cells

2,2,4,4-tetra-brominated diphenyl ether (PBDE-47)—the dominant homologue of polybrominated diphenyl ethers—is a toxic environmental pollutant in the aquatic environment that continuously exists and bioaccumulates in the aquatic food chain. In experimental disease models, melatonin (MEL) has been reported to attenuate necroptosis and inflammatory responses. To further explore the mechanism underlying PBDE-47 toxicity and the mitigative impact of MEL detoxification, in this study, fish kidney cell models of PBDE-47 poisoning and/or MEL treatment were developed. The Ctenopharyngodon idellus kidney (CIK) cell line was treated with PBDE-47 (100 μM) and/or MEL (60 μM) for 24 h. Experimental data suggest that PBDE-47 exposure resulted in the enhancement of cytoplasmic Ca2+ concentration, induction of calcium dysmetabolism, decrease in the miR-140-5p miRNA level, upregulation of Toll-like Receptor 4 (TLR4) and nuclear factor-kappaB (NF-κB), triggering of receptor interacting serine/threonine kinase-induced necroptosis, and NF-κB pathway mediated secretion of inflammatory factors in CIK cells. PBDE-47-induced CIK cell damage could be mitigated by MEL through the regulation of calcium channels and the restoration of disorders of the miR-140-5p/TLR4/NF-κB axis. Overall, MEL relieved PBDE-47-induced necroptosis and the secretion of inflammatory factors through the miR-140-5p/TLR4/NF-κB axis. These findings enrich the current understanding of the toxicological molecular mechanisms of the PBDE-47 as well as the detoxification mechanisms of the MEL.

Eucalyptol relieves imidacloprid-induced autophagy through the miR-451/Cab39/AMPK axis in Ctenopharyngodon idellus kidney cells†.

Imidacloprid (IMI) is a widely used neonicotinoid insecticide that has toxic effects on nontarget organisms. 1,8-Cineole (eucalyptol) is purified from essential oils in several aromatic plants and can prevent xenobiotic toxicity. The kidney is a major organ for xenobiotic elimination and thus has high risk of exposure. The purpose of this research was to clarify the effect of IMI exposure on autophagy in fish kidney cells, determine the potential of eucalyptol to provide cytoprotection from the toxicity of the neonicotinoid pesticide IMI, and identify its mechanism of action. Therefore, the Ctenopharyngodon idellus kidney cell line (CIK cell) was treated with 20 mg/L IMI and/or 20 μM eucalyptol for 48 h as the research objective. The results showed that IMI exposure induced autophagy accompanied by advanced autophagy markers BNIP3, Beclin1 and LC3Ⅱ/Ⅰ in CIK cells, reduced the levels of miR-451, increased the expression of Cab39 and AMPK, inhibited AKT/mTOR signaling, and activated the JNK pathway. Eucalyptol treatment alleviated IMI-induced autophagy and relieved the activation of autophagy-associated signals. These results indicate that eucalyptol could alleviate IMI-induced autophagy through the miR-451/Cab39/AMPK axis in fish kidney cells. These results partly explained the mechanism of biological threat on fish under IMI exposure and the potential application value of EUC in aquaculture.

The endoplasmic reticulum-mitochondrial crosstalk is involved in the mitigation mechanism of eucalyptol on imidacloprid toxicity in Ctenopharyngodon idellus kidney cells

Imidacloprid (IMI), a systemic neonicotinoid insecticide widely used in agriculture, resulting in persistence in aquatic environments that threaten the survival of organisms. Eucalyptol (EUC), a monoterpenoid found in plants, can be applied to medicine, food, and aquaculture. However, the potential protective effects of EUC on cell damage under neonicotinoid pesticide toxicity, and the role of ER stress and its mediated apoptosis and necroptosis in it, remain unclear. Therefore, we treated Ctenopharyngodon idellus kidney (CIK) cells with 20 mg/L IMI and 20 μM EUC for 48 h. The results showed that IMI exposure caused a higher GRP78 levels, activated ATF6, PERK-eIF2α and IRE1-XBP1 pathways, led to the decline of ATPase activities and ATP content, induced the expression of cytokine (TNF-α, IL-1β, IL-6 and INF-γ), triggered BCL2/BAX-mediated apoptosis and RIP1/RIP3/MLKL-dependent necroptosis in the CIK cell line. Surprisingly, EUC had an effect against IMI-induced cytotoxicity, showing that it effectively mitigated the above-mentioned IMI-exposure-induced changes. Taken together, these results suggested that EUC could alleviated IMI-induced cell death and dysimmunity by recovering ER stress/mitochondria imbalance. These results partly explained the mechanism of biological threat on fish under IMI exposure and the potential application value of EUC in aquaculture.

Tea polyphenols alleviates acetochlor-induced apoptosis and necroptosis via ROS/MAPK/NF-κB signaling in Ctenopharyngodon idellus kidney cells.

Overuse of acetochlor pollutes soil and rivers, causing threats to the ecosystem. Studies found that acetochlor exposure could damage multiple organs and tissues in fish and mammal. Tea polyphenols (TP), a natural antioxidant that extracted from tea, has been widely used in food and feed additions. However, the mechanism by which acetochlor causes tissue damage is unclear, and its mitigating agent has yet to be developed. Therefore, we established acetochlor exposure and TP mitigation models by treating Ctenopharyngodon idellus kidney (CIK) cells with 20μM acetochlor and/or 2.5μg/mL TP for 24h, and detected the programmed cell death and its related pathways. The results showed that acetochlor exposure modified antioxidant enzyme activities, induced oxidative stress, resulted in the decline of MMP and ATP levels, enhanced glycolysis and lactate accumulation, and triggered apoptosis and necroptosis in CIK cells. However, TP could inhibit CYP450s expression, activate Nrf2 pathway, enhance antioxidant capacity, further effectively alleviate acetochlor-induced CIK cell death. Overall, the present study proved that acetochlor exposure triggered mitochondrial damage and lactate accumulation-mediated apoptosis and necroptosis through CYP450s/ROS/MAPK/NF-κB pathway. Furthermore, TP could alleviate effectively cell death through relieving oxidative stress and lightening Warburg-like effect.

Melatonin relieves 2,2,4,4-tetrabromodiphenyl ether (BDE-47)-induced apoptosis and mitochondrial dysfunction through the AMPK-Sirt1-PGC-1α axis in fish kidney cells (CIK)

Polybrominated diphenyl ethers (PBDEs) exist in aquatic environments with nephrotoxicity to non-target aquatic species. Melatonin (MT) exhibits an inhibitory effect of oxidative stress and apoptosis in various diseases. 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47) is the main homolog of PBDE samples. Therefore, we investigated the toxic mechanism of BDE-47 and the alleviation effect of MT, the ctenopharyngodon idellus kidney (CIK) cells were treated with BDE-47 (100 μM) and/or MT (60 μM) for 24 h. Firstly, BDE-47 exposure could inhibit oxidative stress-related antioxidant enzymes (T-AOC, SOD, CAT and GPx) and increase the content of malondialdehyde (MDA) to cause oxidative stress. Secondly, BDE-47 enhanced mitochondrial division and inhibited fusion to induce mitochondrial membrane potential in CIK cells. BDE-47 enhanced the mRNA and protein levels of mitochondrial-pathway apoptosis related genes (Cas 3, Cyt-c, and BAX). Thirdly, BDE-47 treatment decreased the expression levels of mitochondrial-related regulatory factors AMPK-Sirt1-PGC-1α signal pathway. Intriguingly, BDE-47-induced oxidative stress, mitochondrial pathway apoptosis and mitochondrial dynamics disorder could be alleviated by MT treatment. Overall, we concluded that MT could relieve BDE-47-induced oxidative stress, mitochondrial dysfunction and apoptosis through the AMPK-Sirt1-PGC-1α axis. These results enrich the mechanisms of BDE-47 poisoning and reveal that MT treatment may be a potential strategy for solving BDE-47 poisoning.

NS38 protein of GCRV 096: Functional analysis and effect on grass carp reovirus replication

Grass carp reovirus (GCRV) seriously affects the grass carp farming, as the causative agent of grass carp hemorrhagic disease. In this study, we found the GCRV 096 NS38 protein contained one conservative PXXP motif similar to σNS of avian reovirus, with very low similarity rates with its homologous GCRV proteins of different genotypes. Furthermore, the soluble GCRV 096 NS38 protein was expressed in Escherichia coli, antiserum was raised and characterised. Addition of purified NS38 protein to culture media of Ctenopharyngodon idella kidney (CIK) cells clearly inhibited replication of GCRV 096 (genotype I); however, replication of GCRV GD108 (genotype Ш) was not significantly affected in CIK cells under the same conditions. It was not possible to induce cross-protection among GCRVs of different genotypes. GCRV multi-genotype should be considered and the NS38 protein was a promising candidate for developing therapies against grass carp hemorrhagic disease. Additionally, bioinformatics analysis suggested that the NS38 protein may be involved in viral genome replication. Keywords: Expression, Functional analysis, Grass carp reovirus 096, NS38 protein, Replication

Functional characterization of two alpha beta hydrolase domain (ABHD) genes associated with lipid accumulation in Ctenopharyngodon idella kidney (CIK) cells

Members of the α/β-hydrolase domain (ABHD) protein family have emerged as novel potential regulators of lipid metabolism and signal transduction. In this study, two ABHD genes (ABHD4 and ABHD5, named CiABHD4 and CiABHD5) were identified and characterized in Ctenopharyngodon idella. The ORF of CiABHD4 cDNA was 1101 bp (MK645600), and encode 366 amino acids. The ORF of CiABHD5 cDNA was 1035 bp (KP325482), and encode 344 amino acids. Phylogenetic analysis showed that the CiABHD4 and CiABHD5 were all closely clustered with the fish ABHD4 and ABHD5 subcluster. Both CiABHD4 and CiABHD5 in Ctenopharyngodon idella were highly expressed in liver. Moreover, we compared the effect of saturated fatty acids (palmitic acid, PA), monounsaturated fatty acids (oleic acid, OA) and polyunsaturated fatty acids (Docosahexaenoic acid, DHA) on CiABHD4 and CiABHD5 expression, and found that PA significantly induce expression of CiABHD4 and CiABHD5, but OA and DHA significantly increased lipid droplet (LD) formation in L8824 and CIK cells. Furthermore, inhibition of ER stress or IRE1α significantly alleviated CiABHD4 and CiABHD5 expression induced by PA, indicating that ER stress or IRE1α pathway was involved in PA-induced CiABHD4 and CiABHD5 expression. Subcellular localization analysis indicated that CiABHD4 and CiABHD5 proteins were differentially distributed in COS7 cells. In addition, the overexpression of CiABHD4 or CiABHD5 increased the significant expression of ATGL genes, and decrease lipid accumulation induced by the OA/PA combination in COS7 cells. We also demonstrated that CiABHD5 does not directly interact with ATGL by Co-IP assay. Taken together, our results suggested that CiABHD4 and CiABHD5 were induced by PA through ER stress, and play essential roles in lipid accumulation by regulating ATGL expression in Ctenopharyngodon idella.

Heme oxygenase-1 protects against inflammatory and apoptosis induced by hemeproteins in Ctenopharyngodon Idellus kidney cells

Infectious bacterial and viral diseases that cause hemolysis are considered life-threatening to grass carp (Ctenopharyngodon idellus), which is a species used in aquaculture worldwide. After heme and hemeproteins (Hbs) are released as a result of hemolysis, pro-inflammatory responses and cell apoptosis occur. Heme oxygenase (HO)-1, a rate-limiting enzyme in heme catabolism, results in the formation of equivalent amounts of biliverdin (BV), carbon monoxide (CO), and ferrous iron (Fe2+). In mammals, many researchers have reported that HO-1 and its metabolites have anti-inflammatory, anti-proliferation, anti-oxidative, and anti-apoptotic effects in the process of maintaining cellular homeostasis. However, the mechanisms underlying the anti-oxidative and anti-apoptotic effects against Hb and heme of teleost HO-1 are poorly understood. To decipher the mechanisms, transcriptomic profiles of the pEGFP-HO-1 or pEGFP-N1 overexpressing in CIK cells after Hb incubation were compared. We show that the mRNA expression levels of pro-inflammatory cytokines, including TNF-α, IL-6, IL-8, IL-1β, and CCL1 were all remarkably down-regulated in the HO-1 overexpressing CIK cells after Hb and heme incubation, while HO-1 over-expression caused a significant increase in the transcriptional level expression of two anti-inflammatory cytokines, IL-10 and TGF-β, by NF-κB inhibition. Further, HO-1 over-expression dramatically induced Nrf2-Keap1 antioxidant signaling pathway-related genes, namely CAT, GSH, SOD, Keap1a, Keap1b, GST, GPX, and GR to eliminate excessive ROS production in CIK cells. Moreover, overexpression of HO-1 significantly inhibited the mRNA expression levels of the following apoptosis-related genes: FasL, Fas, FADD, TNFR1, Apaf1, p53, Bad1, Bad2, Bax, Bid, CytoC, caspase3, caspase8, and caspase9. Conversely, overexpression of HO-1 dramatically activated the mRNA expression levels of anti-apoptosis-related genes, namely Bcl-xl, Bcl2, and Mcl1. These results reveal that overexpression of HO-1 plays a significant role in cell survival by inhibiting Fas, the TNF-α death receptor pathway, and the mitochondrial pathway. In summary, HO-1 overexpression decreases inflammatory cytokine secretion and down-regulated the expression of pro-apoptotic genes and the generation of ROS may both depend on NF-κB, Fas, TNF-α death receptor and mitochondrial pathway inhibition, Nrf2-Keap1 as well as p38 MAPKs activation.

Hemeprotein amplifies the innate immune receptors of Ctenopharyngodon idellus kidney cells through NF-κB- and MAPK-dependent reactive oxygen species generation

Infectious bacterial and viral diseases that cause hemolysis are considered life-threatening to grass carp (Ctenopharyngodon idellus), which is a species used in aquaculture worldwide. After heme and hemeproteins (Hb) are released as a result of hemolysis, the effect of excess Hb and heme on tissues remains to be characterized. To decipher the mechanisms, after incubation with Hb, we showed that lipopolysaccharide (LPS), Hb, and heme increased the cytotoxicity and secretion of inflammatory cytokines such as interleukin (IL)-6, chemokine (C-C motif) ligand 1 (CCL1), tumor necrosis factor (TNF)-α, IL-6, and IL-1β in vitro, which was due to stimulation of the expression of innate immune receptors, such as nucleotide-binding oligomerization domain (NOD2), toll-like receptor 2 (TLR2), TLR 4, and TLR3. The formation of reactive oxygen species (ROS) and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB were important for increasing the cytokine production to induce heme, Hb, and LPS. Moreover, we confirmed that after LPS, Hb, and heme challenge, superoxide dismutase (SOD) and glutathione (GSH) synthetase (GSS) also caused remarkable destruction. However, catalase (CAT) and heme oxygenase-1 (HO-1) were strongly activated. In summary, our research findings present a framework through which heme and Hb concentrations amplify the secretions of inflammatory cytokines, which are induced by pattern recognition receptor (PRR) activation and present possible paths for immune intervention during infection with viral diseases and hemolytic bacterial.

Characterization of SR-B2a and SR-B2b genes and their ability to promote GCRV infection in grass carp (Ctenopharyngodon idellus)

Scavenger receptor class B type 2 (SR-B2) is a pattern recognition receptor involved in innate immunity in mammals; however, the immunological function of SR-Bs in fish remains unclear. In this study, the full-length cDNA sequences of SR-B2a and SR-B2b from grass carp (Ctenopharyngodon idellus) were cloned and designated as CiSR-B2a and CiSR-B2b. Multiple alignments and phylogenetic analyses deduced that CiSR-B2a and CiSR-B2b had the highest evolutionary conservation and were closely related to the zebrafish (Danio rerio) homologs, DrSR-B2a and DrSR-B2b, respectively. Both CiSR-B2a and CiSR-B2b were expressed in all the tested tissues, with the highest expression levels found in the hepatopancreas. In Ctenopharyngodon idellus kidney cells (CIK), CiSR-B2a and CiSR-B2b were mainly located in the cytoplasm, and a small amount located on the plasma membrane. After challenge with Grass Carp Reovirus (GCRV), the expression of CiSR-B2a and CiSR-B2b were significantly upregulated in the spleen (about 10.27 and 27.19 times higher than that at 0 day, p < 0.01). With CiSR-B2a or CiSR-B2b overexpressed in CIK, the relative copy number of GCRV in the cells was both significantly increased compared to that in the control group, indicating that CiSR-B2a and CiSR-B2b may be important proteins during the infection processes of GCRV.

ATF6-DGAT pathway is involved in TLR7-induced innate immune response in Ctenopharyngodon idellus kidney cells

DGAT1 and DGAT2 are two acyl-CoA:diacylglycerol O-acyltransferase (DGAT) enzymes that catalyze the final step in triglyceride (TG) synthesis. TGs are the primary constituents of lipid droplets (LDs). Although it has been demonstrated that LDs modulate immune and inflammatory responses in CIK cells, little is known about whether DGAT1 and DGAT2 involve in this process. Firstly, grass carp DGAT2 was isolated and characterized, encoding 361 amino acids, and all DGAT2 proteins in genomic structures are conserved in vertebrates. Then, using TLR7 agonist, we induced LDs accumulation in CIK cells. Only DGAT1b and DGAT2 were upregulated in forming TLR7 agonist induced-LDs. Next, we utilized small-molecule inhibitors of DGAT1 and DGAT2. The results indicated that DGAT1 inactivation attenuated TG content and the relative expressions of IFNα3, NF-κB, IL-1β, and TNFα genes, whereas DGAT2 inhibition decreased TG content and the relative expressions of MyD88, IRF7, IFNα3, NF-κB, IL-1β, and TNFα genes, implying that DGAT1-generated LDs and DGAT2-generated LDs contribute to TLR7-induced immune response via different signaling pathways. Finally, inhibiting ATF6 effectively decreased DGAT-generated LDs accumulation and the expression of TLR7 signaling-related genes induced by TLR7 agonist, implying that ATF6 UPR pathway may mediate the role of DGAT-generated LDs in TLR7 signaling. Overall, we demonstrate that DGAT1 and DGAT2-catalyzed TAG synthesis may generate different LDs to provide distinct signaling platforms for innate TLR7 signaling.

New insights into crosstalk between apoptosis and necroptosis co-induced by chlorothalonil and imidacloprid in Ctenopharyngodon idellus kidney cells.

Overuse and co-exposure of pesticides have become a public health problem and threat seriously water health and environmental organisms and even humans. Chlorothalonil (CT) and imidacloprid (IMI) are high-selling pesticides worldwide, which can persist in the environment, and present a series of severely toxic effects on non-target animals. However, the effect of co-application on aquatic organisms is unknown. Based on the concept of the toxic unit (TU), toxic interaction of CT and IMI was evaluated and showed the additive and synergistic toxicity on Ctenopharyngodon idellus (grass carp) kidney cell line (CIK cells). Cell death analysis found an obvious increase of the apoptosis and necrosis rates exposed to CT and IMI, and aggravation when applied together. Moreover, CT and IMI co-exposure accelerated the inhibition of CYP450s/ROS/HIF-1α signal, the decline of energy metabolism, mitochondrial dynamics disorder, activation of Bcl2/Bax/Cyt C/Casp3/Casp9 pathway and RIP1/RIP3/MLKL pathway. Bioinformatics analysis showed autophagy, cell response, NOD-like receptor signaling pathway might be affected by co-exposure. In summary, the above results indicate that co-exposure to CT and IMI has synergistic toxicity and aggravates cell death via inhibition of the CYP450s/ROS/HIF-1α signal. These data provide new insights for evaluating the stacking interaction and revealing the toxicological effects of pesticide mixture.