1. Development of novel tetra- and nucleotide micro satellite markers for giant grouper Epimetheus lanceolatus using 454 pyrosequencing. Giant grouper (Epimetheus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable.
This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and nucleotide micro satellite markers in giant grouper from Sarah, Malaysia.
Of these loci, 16 had tetra- and 8 had nucleotide repeats, all of which exhibited polymorphisms within easily genotype regions. In general, TLS have two downstream signaling pathways, the eyelid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon (Trip)-dependent pathways, which lead to the activation of nuclear factor-kappa B (NFL) and interferon regulatory factor 3 (IRF3).
3. In Vito activity of (-)-deoxypergularinine, on its own and in combination with anti-tubercular drugs, against resistant strains of Mycobacterium tuberculosis. 4. Schisandrin B inhibits Th1/Th17 differentiation and promotes regulatory T cell expansion in mouse lymphocytes.
Chen Z1, Go M2, Song G2, GAO J2, Zhang Y2, Jing Z2, Liu T2, Dong C3. Schisandrin B (Sch-B), the most abundant active ingredient of the fruit of Chandra Chinese, has been proposed to have antioxidant, anti-tumor and anti-inflammatory effects.
The present study was undertaken to investigate the effect of Sch-B on differentiation of T helper cells (Th). Using mouse splenic lymphocytes stimulated with concanavalin A (Con A) in vitro and ex Vito as inflammation models, we found that Sch-B significantly inhibited secretion of Th1 and Th17 related cytokines, such as IFN and IL-17.
The full-length EcMDA5 CDA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homology from rock bream (Oplegnathus fascists). The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced idiopathic effect (CPE) progression and significantly inhibited viral gene transcription of Ronny and SGI.
Moreover, over expression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (Ire) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. If you log out, you will be required to enter your username and password the next time you visit.
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2 College of Fisheries and Life Science, Hainan Tropical Ocean University, No. 2 College of Fisheries and Life Science, Hainan Tropical Ocean University, No.
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bright field (A) and fluorescent image (B) of EPC cells transected with pcDNA3.1-EGFP empty plasmid.
Bright field (C) and fluorescent image (D) of EPC cells transected with pcDNA3.1-EGFP- Cc IRF10 plasmid. Data Availability Statement Interferon (IFN) regulatory factors (IRS), as transcriptional regulatory factors, play important roles in regulating the expression of type I IFN and IFN- stimulated genes (Isis) in innate immune responses.
In addition, they participate in cell growth and development and regulate oncogene sis. In the present study, the CDA sequence of IRF10 in common carp (Cyprus cardio L.) was characterized (abbreviation, Cc IRF10).
The predicted protein sequence of Cc IRF10 shared 52.7–89.2% identity with other telecast IRF10s and contained a DNA-binding domain (DBD), a nuclear localization signal (NLS) and an IRF-associated domain (IAD). Phylogenetic analysis showed that Cc IRF10 had the closest relationship with IRF10 of Ctenopharyngodon Della.
Cc IRF10 expression was unregulated in the spleen, head kidney, fore gut and hind gut upon polyinosinic:polycytidylic acid (poly I:C) and Aeromonas hydrofoil stimulation and induced by poly I:C, lipopolysaccharide (LPS) and peptidoglycan (PGN) in peripheral blood leukocytes (PBL) and head kidney leukocytes (Hals) of C. cardio. In addition, over expression of Cc IRF10 was able to decrease the expression of the IFN and IFN-stimulated genes Per and ISG15.
These results indicate that Cc IRF10 participates in antiviral and antibacterial immunity and negatively regulates the IFN response, which provides new insights into the IFN system of C. cardio. The online version contains supplementary material available at 10.1186/s12917-020-02674-z.
Keywords: Common carp (Cyprus cardio L.), Interferon regulatory factor 10 (IRF10), poly I:C, Aeromonas hydrofoil, IFN response Interferon (IFN) regulatory factors (IRS), as transcriptional regulatory factors, play multiple important roles in host immune responses and other physiological processes; for example, they regulate the expression of type I IFN and IFN-stimulated genes (Isis) , the activation and differentiation of distinct immune cell populations, cell growth, differentiation and apoptosis .
The N-terminal regions of IRS share a highly conserved DNA-binding domain (DBD), which contains five or six tryptophan repeats . To date, 11 kinds of IRS have been identified in vertebrates , which can be divided into four subfamilies, IRF1 (IRF1, 2, and 11), IRF3 (IRF3 and 7), IRF4 (IRF4, 8, 9, and 10) and IRF5 (IRF5 and 6), according to their differences in the C-terminal region .
Phylogenetic analysis has shown that GG IRF10 clusters into the IRF4 subfamily and plays a crucial role in the later stages of the immune response to invading pathogens in G. Gallup . Functional characterization of fish IRF10 has also begun recently, and this gene has been identified in telecasts including zebrafish (Dario period) , orange spotted grouper (Epimetheus coincides) , Japanese flounder (Paralichthys Olivares) , Atlantic cod (Gads Joshua) , grass carp (Ctenopharyngodon Della) , rainbow trout (Oncorhynchus my kiss) , Asian swamp eel (Monsters Albus) , mandarin fish (Sniper cheats) , Senegalese sole (Sole Senegalese) and blunt snout bream (Melodrama amblycephala) .
The telecast IRF10 genes are constitutive expressed in a variety of tissues and can be unregulated by the viral mimic polyinosinic:polycytidylic acid (poly I:C) , lipopolysaccharide (LPS) , viruses or bacteria , suggesting that IRF10 plays roles in immune responses to both viral and bacterial infections. Furthermore, in zebrafish and orange spotted grouper, IRF10 inhibits IFN1 and IFN3 promoter activation and negatively regulates fish antiviral gene expression to prevent an excessive immune response, which is a unique regulatory mechanism of IFN responses in telecasts .
As a pivotal aquaculture fish species widely cultured in both Asia and Europe, common carp (Cyprus cardio L.) is also a good model for exploring the immune system of telecasts . In this study, we identified the full-length CDA sequence of IRF10 in C. cardio (named Cc IRF10) and investigated its function.
We examined the tissue distribution of Cc IRF10 in healthy common carp and then evaluated the expression level of Cc IRF10 upon viral or bacterial stimulation both in vivo and in vitro to determine its function in the immune response against pathogens. Furthermore, the regulatory role of Cc IRF10 in the IFN signalling pathway was also determined in this study.
The results will contribute to understanding of the innate immune system of fish. C. Carpio specimens were purchased from a local fish farm (Jinan, Shandong, China), selected for size (approximately 200g per fish), maintained in recirculating tap water at 20 °C and fed daily for more than 1 week before challenge and sampling.
After treatment, the fish were euthanized by immersion in a solution of tricking methane sulfonate (MS222, Sigma-Aldrich) at a concentration of 100 mg/l of water. The primers were designed based on the known fish IRF10 CDA sequences.
Then, 3 and 5 Full RACE Core Sets (Tamara) were used to obtain the full-length CDA sequence. The PCR products were ligated into the pMD18-T vector (Tamara) and transformed into competent E. coli DH-5 for sequencing (Nitrogen).
The domains of the protein sequence were analyzed using the Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de). Multiple alignment and phylogenetic analysis were performed using MEGA 5.0.
Poly I:C and A. hydrofoil challenge experiments were performed in C. cardio as previously described . In the A. hydrofoil challenge experiment, the bacteria were inactivated in 0.5% formalin at 37 °C for 36h and then resuspended in PBS.
Then, 50 fish were intraperitoneally injected with 500l of A. hydrofoil (at a dose of 2.0×10 8 cells). At 0, 3, 6, 12, 24, 48 and 72h post injection (hip), the spleen, head kidney, fore gut and hind gut were collected from three fish at every time points (n =3).
Peripheral blood leukocytes (PBL) and head kidney leukocytes (Hals) were isolated from C. cardio according to a previous report . In brief, C. cardio peripheral blood and head kidney cell suspensions were loaded onto freshly prepared 34%/51% Per coll (Sigma) density gradients and separated via gentrification at 650× g for 30min.
The cells were resuspended in cold Leibniz’s L-15 medium with 10% fetal bovine serum (FBS), 100 UI/ml penicillin and 100 mg/ml streptomycin. PBL or Hals (1×10 6) were maintained in a 24-well cell culture plate with 500l in each well and treated with 5l poly I:C (500g/ml), LPS (1 mg/ml) or peptidoglycan (PGN, 1 mg/ml).
At 0, 3, 6, 12 and 24h post stimulation, triplicate cell samples were harvested, and total RNA was extracted (n =3). The Of of Cc IRF10 was amplified using Fusion High-Fidelity DNA polymerase (Prime STAR) with specific primers.
Purified fragments were digested with the Said and Eco RI restriction enzymes, ligated into the pcDNA3.1-EGFP vector, and transformed into E. coli Top10 cells. The over expression vector pcDNA3.1-EGFP- Cc IRF10 (abbreviation, pcIRF10) was verified by sequencing.
Epithelium populous Cypriot (EPC) cells were seeded in 24-well plates with 500l in each well at a concentration of 4×10 5 cells/ml and maintained at 25 °C in M199 medium (Cyclone) supplemented with 10% FBS, 100U/ml penicillin and 100g/ml streptomycin (Rico) for 1 day so that they reached approximately 80% confluence. EPC cells transected with empty plasmids served as controls, and the gene expression of IFN, Per, ISG15 and TNF was detected after over expression of Cc IRF10.
Comparisons between the experimental group and the control group were performed using one-way analysis of variance (ANOVA) in Grafted Prism 5, and a value of P <0.05 was considered to indicate significance. MT646905) contains a 78bp 5-untranslated region (UTC), a 26bp 3but containing mRNA instability motifs (1244 AAAA 1249), and an Of of 1170bp that translates into a 390-amino acid putative peptide with a predicted molecular mass of 44.4kDa.
The expression patterns of Cc IRF10 in 11 tissues of healthy common carp, including the liver, spleen, head kidney, fore gut, hind gut, gills, gonad, skin, muscle, burial epithelium and brain, were detected by real-time PCR. Cc IRF10 mRNA expression in the liver, spleen, head kidney, gills, skin, fore gut, hind gut, burial epithelium, gonad, muscle and brain was determined by real-time PCR.
The gene expression levels were normalized using 40S ribosomal protein S11 mRNA. Upon poly, I:C stimulation, the peak expression of Cc IRF10 appeared at 6 hip in the spleen (4.5-fold, P <0.05), fore gut (27.5-fold, P <0.05) and hind gut (7.5-fold, P <0.05).
In the head kidney, Cc IRF10 expression peaked at 3 hip (7.5-fold, P <0.05) (Fig. Expression analysis of Cc IRF10 in response to poly I:C challenge in vivo.
Total RNA was extracted from spleen (a), head kidney (b), fore gut (c) and hind gut (d) tissues at 0 (as a control), 3, 6, 12, 24, 48 and 72h post injection for real-time PCR. In A. hydrofoil -infected fish, Cc IRF10 was unregulated in the head kidney (6.8-fold, P <0.05), fore gut (13.7-fold, P <0.05) and hind gut (3.0-fold, P <0.05) at 6 hip.
In the spleen, Cc IRF10 reached its peak at 48 hip, with a 4.6-fold induction (P <0.05) (Fig. Expression analysis of Cc IRF10 in response to A. hydrofoil challenge in vivo.
Total RNA was extracted from spleen (a), head kidney (b), fore gut (c) and hind gut (d) tissues at 0 (as a control), 3, 6, 12, 24, 48 and 72h post injection for real-time PCR. To examine the Cc IRF10 transcription level in response to poly I:C, LPS or PGN challenge in vitro, we isolated leukocytes from the peripheral blood and head kidneys of C. cardio.
A, real-time PCR data showed that the expression of Cc IRF10 in the PBL was unregulated by poly I:C (2.0-fold, P <0.05) and PGN (2.2-fold, P <0.05) at 12h, but not by LPS. Expression levels of Cc IRF10 in PBL (a) and Hals (b) upon poly I:C and LPS stimulation.
Cells were collected at 0 (as a control), 3, 6, 12 and 24h post-infection for RNA extraction and real-time PCR analysis. To investigate the regulatory role of Cc IRF10 in the IFN signalling pathway, the gene expression of IFN, two IFN-stimulated genes (Per and ISG15) and TNF was detected after over expression of Cc IRF10 in EPC cells (Fig.
Relative expression of IFN (a), Per (b), ISG15 (c) and TNF (d) in Cc IRF10-transfected EPC cells. IRF10, which has been reported to play important roles in immune responses to both viral and bacterial infections in telecasts, can inhibit the activation of IFN promoters and negatively regulate fish antiviral gene expression to prevent an excessive immune response .
In the present study, a non-mammalian IRF10 gene was cloned from common carp, and the antiviral and antibacterial immune functions of Cc IRF10 were investigated. Notably, the five well-conserved tryptophan residues in D. period, C. Della, G. Gallup, and C. cardio are all at positions 10, 25, 37, 57 and 76 in the N-terminus.
The IAD, which is responsible for homo/heterodimer interactions of the IRS and association with other transcription factors, is less conserved than the DBD . Phylogenetic analysis of predicted IRF10 protein sequences of C. cardio and other vertebrate species supported the division of IRF10 into two branches: telecast and bird.
These results match the established evolutionary relationships among telecast and other vertebrate species and support the authenticity of the nomenclature for IRF10. This ubiquitous tissue expression pattern supports the findings of previous studies on IRS in telecasts, including M. Albus , G. Joshua , D. period , O. my kiss , turbot (Scophthalmus Maximus) , P. Olivares , rock bream (Oplegnathus fascists) , blunt snout bream (Melodrama amblycephala) and half-smooth tongue sole (Colossus semibreves) .
In P. Olivares, the expression of IRF10 has been found to be very high in the gills, intestine, trunk kidney, heart, stomach, head kidney and PBL; in C. Della, IRF10 expression has been found to be high in all tested tissues, with the highest expression in the thymus and gills; and in M. Albus, the highest expression level has been observed in intestine, whereas the lowest level has been found in the liver . Previous studies on C. cardio have shown that the expression of IRF1, IRF3, IRF5 and IRF7 is unregulated upon stimulation with poly I:C or viruses .
In the present study, following poly I:C injection, the induction of Cc IRF10 in the fore gut (27.5-fold) was much stronger than that in the other tissues (4.5- to 7.5-fold), revealing the important role of Cc IRF10 in the mucosal immune system response to poly I:C (Fig. Moreover, similar results were observed in PBL and Hals with poly I:C stimulation (Fig.
The observed induction of IRF10 expression by various viruses and poly I:C suggests that the fish IRF10 may play a crucial role in protecting the host from viral infection. A. Hydrophila, a well-known fish-pathogenic bacterium, is primarily found in temperate and freshwater environments and causes infections in various organisms.
Moreover, A. hydrofoil breakouts have caused great economic losses around the world . When fish were challenged with A. hydrofoil, the levels of Cc IRF10 were unregulated in all four tissues, with the highest induction in the fore gut (Fig.
Upon Aeromonas salmonicida infection, the greatest expression of the long isoform of G. Joshua IRF10 occurs at 24 hip, whereas the highest expression of the short isoform is observed at 6 hip at 10 °C and 16 °C, suggesting the distinct roles of the two isoforms in the immune system of G. Joshua . Therefore, it was unsurprising that Cc IRF10 was not unregulated by LPS in the PBL (Fig.
PGN, a major component of the bacterial cell wall, consists of sugars and amino acids. Similar to the findings of a previous study regarding head kidney macrophages of O. my kiss, Cc IRF10 was unregulated by PGN stimulation in vitro .
Hence, our in vivo and in vitro findings, together with the previous analogous results, suggest that Cc IRF10 is more susceptible to poly I:C than to A. hydrofoil infection and plays a substantial role in the fore gut, which is a mucosal immune organ. Moreover, fish IRF10 may play essential roles in both antiviral and antibacterial defense, as reported for G. Gallup IRF10.
In mammals, Ions are natural glycoproteins produced by cells of the innate and adaptive immune systems in most vertebrates in response to challenge by viruses, bacteria, fungi, parasites, and tumor cells . Similarly, in fish, Ions play a crucial role in innate immunity .
To investigate the regulatory role of Cc IRF10 in the IFN response, we detected the mRNA expression of type I IFN, Isis (Per and ISG15) and TNF in Cc IRF10-transfected EPC cells. The transcription of IFN, Per, ISG15 was down regulated in EPC cells transected with the pcDNA3.1- Cc IRF10 plasmid (Fig.
This result is in line with the findings of a previous study that over expression of D. period IRF10 down regulated the expression of IFN-stimulated genes induced by poly I:C and promoted the replication of spring viremia of carp virus (SVC) in EPC cells . Moreover, this study found that the Ire site in the promoter was responsible for Dr IRF10-mediated inhibition of gene expression of Ions and Isis .
The mechanism involved in the inhibition of IFN signalling pathway in common carp may be similar, which needs our further study. However, G. Gallup IRF10 can up regulate the expression of MHC class I and GBP , suggesting that the function of IRF10 might be different between fish and birds.
In the present study, the full-length CDA sequence of IRF10 from common carp was identified and characterized. In vivo and in vitro studies indicated that Cc IRF10 participates in both antiviral and antibacterial immune responses.
The study will provide a valuable experimental foundation for future studies on the immune system of common carp and a theoretical basis for the prevention of fish disease. Bright field (A) and fluorescent image (B) of EPC cells transected with pcDNA3.1-EGFP empty plasmid.
YYZ, SAS, HP and HW performed the experiments. The funders were not involved in the study design; collection, analysis and interpretation of the data; or in writing the manuscript.
The protocol was approved by the Animal Experimental Ethics Committee of Shandong Normal University (Permit Number: AEECSDNU2017007). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Taobao AHU and Shipman Shan contribute equally to this work. Ma mane Y, Halterneck C, Lenin P, Alga rte M, Servant MJ, Le Page C, Delta C, Won H, Lin R, Scott J. Interferon regulatory factors: the next generation.
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