Stimulation Of Poly Research Articles

Two cytokine receptor family B (CRFB) members in orange-spotted grouper Epinephelus coioides, EcCRFB3 and EcCRFB4, negatively regulate interferon immune responses to assist nervous necrosis virus replication

Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 990 bp encoding 329 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I-induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFNRs to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV.

IKKε positively regulates NF-κB, MAPK, and IRF3-mediated type I IFN signaling pathways in Japanese eel (Anguilla japonica)

IKKε is an IκB kinase participating in the control of NF-κB and type I IFN signal pathways in mammals. However, the function of IKKε in regulating immune response is largely unknown in teleost. Herein, an IKKε homologue named AjIKKε was characterized in Japanese eel (Anguilla japonica). AjIKKε has an N-terminal kinase domain, a ubiquitin-like domain, and a coiled coil-containing domain (CC), which is conserved and similar to its counterpart in mammals. Expression analysis showed that AjIKKε could be up-regulated in kidney, spleen, and particularly in liver under the stimulation of poly I:C, LPS, and Aeromonas hydrophila infection. In vitro, the mRNA levels of AjIKKε were significantly provoked in eel liver cells stimulated by LPS and poly I:C, or the different concentrations of A. hydrophila. The overexpression of AjIKKε could not only induce a significantly higher level of promoter activity of human NF-κB, AP-1, and IFN-β in a dose-dependent manner but also up-regulate the activation of promoters of Japanese eel cRel, AP1, IL6, IFN4, IRF3, and IRF7 in HEK293 cells. RNAi studies showed that after AjIKKε was knocked down, the expression levels of IL1, IL6, TNFα, c-Jun, IFN2, IFN3, MX1, MX2, and IRF3 genes were significantly down-regulated in liver, spleen, and kidney of Japanese eels. In addition, the mutants of AjIKKε-K39A, AjIKKε-S174A, and AjIKKε-ΔCC failed to activate Japanese eel IFN4, IRF3 and human IFN-β promoters in HEK293 cells. Collectively, these results suggest that AjIKKε may function as a positive regulator of NF-κB, MAPK, and IRF3-mediated type I IFN signaling pathways related to immune response evoked by bacterial and viral infection.

Circular RNA circPlce1 regulates innate immune response in miiuy croaker, Miichthys miiuy

In recent years, more and more researchers have devoted to the study of circular RNAs (circRNAs) in noncoding RNAs. As an important regulator in a variety of biological processes, circRNAs are relatively abundant in the study of mammals, while research in lower vertebrates is still lacking. In this study, we found a circRNA, circPlce1, related to innate immune response in Miichthys miiuy (miiuy croaker). The experimental results confirmed that circPlce1 could promote the production of antiviral genes and inflammatory response under the stimulation of poly (I: C) and LPS. We also confirmed that circPlce1 can promote NF-κB and IRF3 pathways through luciferase reporter assay experiment. In addition, we also found that circPlce1 can promote cell proliferation and improve cell viability. In conclusion, our results showed that circPlce1 plays an active role in regulating inflammatory response, cell proliferation and cell viability, providing a foundation for the study of the biological function of circRNAs in the innate immune response in teleost fish.

Molecular cloning and functional characterization of HMGB1 and HMGB2 in large yellow croaker Larimichthys crocea

High mobility group box 1 (HMGB1) and HMGB2 have been demonstrated to be key regulators not only in DNA recombination, replication, gene transcription, but also in host inflammation and immune responses. In the present study, orthologs of HMGB1 and HMGB2 named Lc-HMGB1 and Lc-HMGB2 were characterized in large yellow croaker (Larimichthys crocea). The ORFs of Lc-HMGB1 and Lc-HMGB2 are 621 bp and 648 bp, encoding proteins of 206 aa and 215 aa, with the putative Lc-HMGB1 and Lc-HMGB2 proteins both contain two HMG domains, respectively. The genome organizations of Lc-HMGB1 and Lc-HMGB2 are both composed of four exons and three introns, which are conserved in vertebrates. Lc-HMGB1 and Lc-HMGB2 were identified as cell nucleus localized proteins, and were ubiquitously distributed in the examined organs/tissues. Additionally, Lc-HMGB1 was significantly up-regulated under LPS and PGN stimulation, whereas the stimulation of poly I:C, LPS, PGN, and Pseudomonas plecoglossicida infection could significantly induce Lc-HMGB2 expression in vivo. Notably, both Lc-HMGB1 and Lc-HMGB2 overexpression could significantly up-regulated the expression of diverse immune-related genes, including IFN1, IRF3, ISG15, ISG56, RSAD2, g-type lysozyme, and TNF-α. Moreover, overexpression of Lc-HMGB1 could also induce the expression of IRF7 and Mx. These results collectively indicate that Lc-HMGB1 and Lc-HMGB2 play important roles in host immune responses against pathogen infection.

Interferon-Induced Protein 6-16 (IFI6-16) from Litopenaeus vannamei Regulate Antiviral Immunity via Apoptosis-Related Genes.

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)–Vago–JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF–IFN–JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT–IFI6-16–caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.

Differential activation of chicken gamma delta T cells from different tissues by Toll-like receptor 3 or 21 ligands

Gamma delta (γδ) T cells are highly enriched in mucosal barrier sites including intestinal tissues where microbial infections and tumors often originate in mammals. Human γδ T cells recognize stress antigens and microbial signals via their T cell receptor (TCR), natural killer (NK) receptors, and pattern recognition receptors. However, little is known about antigens or ligands capable of stimulating chicken γδ T cells. The results of the present study demonstrated that polyinosinic-polycytidylic acid (poly(I:C)), a Toll-like receptor (TLR)3 ligand, significantly induced upregulation of CD8α molecules on circulating and lung γδ T cells. Moreover, poly(I:C) stimulation induced interferon (IFN)-γ production from splenic and lung CD8α+ γδ T cells while Cytosine-phosphate-Guanine oligodeoxynucleotides (CpG-ODN) 2007, a TLR21 ligand, stimulation induced IFN-γ production by circulating γδ T cells. Neither poly(I:C) nor CpG-ODN 2007 stimulation elicited degranulation of γδ T cells. Additionally, the results revealed that CpG-ODN 2007 induced IFN-γ production from TCR-stimulated γδ T cells sorted from spleen. In our experiments, isopentenyl pyrophosphate (IPP), 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), or zoledronate (Zol) stimulation did not induce IFN-γ production or degranulation in γδ T cells. Taken together, a combination of CpG-ODN 2007 and anti-CD3ε monoclonal antibodies (mAbs) can stimulate chicken γδ T cells and induce production of IFN-γ by these cells while IFN-γ production by γδ T cells induced by stimulation of poly(I:C) needs signals from other cells. These results suggest that chicken γδ T cells can sense invading pathogens via TLRs and produce IFN-γ as a first line of defense.

Molecular characterization, expression patterns, and subcellular localization of a classical and a novel nonclassical MHC class I α molecules from Japanese eel Anguilla japonica

Major histocompatibility complex (MHC) molecules play a critical role in the immune response of vertebrate animals by presenting foreign antigens to T lymphocytes. In this study, we first cloned and identified a classical and a novel nonclassical MHC I α molecules from Japanese eel (Anguilla japonica), named as AjMHC I-UBA and AjMHC I-L, respectively. The full-length cDNA of AjMHC I-UBA contains an open reading frame (ORF) of 1047 bp encoding a predicted protein of 348 amino acids, while AjMHC I-L 1089 bp encodes 362 amino acids. The multiple alignment of the amino acid sequence showed that AjMHC I-UBA and AjMHC I-L consist of an N-terminal MHC I superfamily domain within α1 and α2 helices, an IgC-MHC I α3 domain, and a transmembrane region close to the C-terminal, which are similar to other fish and mammal species. Molecular polymorphism analysis showed that eight different major alleles of AjMHC I-UBA, named as AjMHC I-UBA*0101～1001, were identified from six Japanese eel individuals. Furthermore, a distinguishing signature of the nonclassical L-lineage genes specific motif “HINMTL”, including an N-glycosylation site (NXS/T), was present in the α3 domain of AjMHC I-L sequences. Although the predicted three-dimensional structures of AjMHC I-UBA and AjMHC I-L are similar to that of human MHC I α, phylogenetic analysis showed that these two protein molecules belong to classical MHC I UBA gene of U-lineage and nonclassical MHC I gene of L-lineage, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the highest expression of AjMHC I-UBA and AjMHC I-L was found in the intestine and the expression level of AjMHC I-UBA in all of the examined tissues was significantly higher than that of AjMHC I-L. The expressions of AjMHC I-UBA and AjMHC I-L in liver, kidney and spleen were significantly induced following injection with the viral mimic poly I:C, LPS, and Aeromonas hydrophila infection. In vitro, the AjMHC I-UBA and AjMHC I-L transcripts of Japanese eel liver cells were significantly enhanced by the treatment of poly I:C or the stimulation of the high concentration of A. hydrophila. Subcellular localization showed that under natural conditions, AjMHC I-UBA and AjMHC I-L were uniformly distributed in the cytoplasm, and aggregated into spots or flakes in the cytoplasm after the stimulation of poly I:C or LPS. These results collectively suggested that AjMHC I-UBA and AjMHC I-L are important components possibly involved in Japanese eel defense against viral and bacterial infection. Taken together, these findings provide valuable insight into the immune function of MHC class I in the immune system of teleost.

Identification and characterization of CD152 in Nile tilapia (Oreochromis niloticus) playing roles in host immune response against bacterial pathogens

The CD152, as named CTLA-4 (Cytotoxic T lymphocyte-associated antigen-4), is an important costimulatory molecule on the surface of T cells and plays an essential role in the cell-mediated immune response. In this work, Nile tilapia (Oreochromis niloticus) CD152 (OnCD152) was successfully cloned, and its characterization in response to bacterial pathogens and interaction with its ligand OnCD80/86 was also investigated. The open reading frame (ORF) of OnCD152 was 705 bp, encoding a polypeptide with 234 amino acids. The OnCD152 protein shared some homology with other known CD152 molecule from teleost species, containing conservative key sites, namely YPPPY, GNGT and four cysteine residues (Cys 45, Cys 97, Cys 123, Cys 151). The mRNA level of OnCD152 was extensively expressed in the collected healthy tissues. Following the stimulation of Poly (I:C), Streptococcus agalactiae and Aeromonas hydrophila, CD152 was significantly upregulated in the head kidney and spleen. The transcriptional upregulations of OnCD152 were also detected in Nile tilapia leukocytes after in vitro stimulations with Poly (I:C), S. agalactiae and A. hydrophila. In addition, the recombinant protein (r)OnCD152 was purified, and its binding ability to (r)CD80/86 strengthen with the increase of concentration. Taken together, this study demonstrated that OnCD152 was likely involved in host immune response against bacterial pathogens. Moreover, this research first cover about the capacity of OnCD152 binding with ligand CD80/86 in Nile tilapia (Oreochromis niloticus), which enrich the understanding for exploring the CD152 function in immune response in teleost fish in the future.

Tilapia dsRNA-activated protein kinase R (PKR): An interferon-induced antiviral effector with translation inhibition activity

The dsRNA-activated protein kinase R (PKR) is one of key antiviral effectors induced by interferons (IFNs), and its functions are largely unknown in tilapia, an important commercial fish species suffering from several viral infectious diseases. In the present study, a PKR gene named On-PKR was identified and cloned from Nile tilapia, Oreochromis niloticus. On-PKR gene was constitutively expressed in all tissues examined, with the highest expression level observed in head kidney and liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). Importantly, the expression of On-PKR is induced by group I and group II IFNs with distinct induction kinetics in vivo: group I IFN elicits a relative delayed but sustained induction of On-PKR, whereas group II IFN triggers a rapid and transient expression of On-PKR. Moreover, the overexpression of On-PKR has been proven to inhibit the protein translation and virus replication in fish cells. The present study thus contributes to a better understanding of the functions of antiviral effectors in tilapia, and may provide clues for the prevention and therapy of viral diseases in fish.

Expression and antibacterial analysis of galectin-8 and -9 genes in mandarin fish, Siniperca chuatsi

Galectin-8 and galectin-9 belong to tandem repeat-type galectins, and in the present study, these two genes were cloned in mandarin fish Siniperca chuatsi. The open reading frame (ORF) of the mandarin fish galectin-8 and galectin-9 contains 942, and 1008 bp, encoding 313 and 335 amino acids, respectively. As a conserved feature, an N-terminal carbohydrate recognition domain (CRD), and a C-terminal CRD were observed in each of the two galectins in mandarin fish. In healthy fish, galectin-8 and -9 were constitutively expressed in all organs/tissues examined, and their expression can be induced following the stimulation of LPS and poly(I:C). It is obvious that galectin-8 had a higher increase at mRNA level following the stimulation of poly(I:C). It is further demonstrated that mandarin fish galectin-8 inhibited the growth of Flavobacterium columnare and Streptococcus agalactiae, and in addition to the two species of bacteria, galectin-9 inhibited also the growth of Edwardsiella piscicida, which provides the basis for further understanding their antibacterial role in immune response of mandarin fish.

Identification and characterization of tilapia CRFB1, CRFB2 and CRFB5 reveals preferential receptor usage of three IFN subtypes in perciform fishes

Type I interferons are a subset of cytokines playing central roles in host antiviral defense, and their effects depend on the interaction with the heterodimeric receptor complex. Surprisingly, two pairs of the receptor subunits, CRFB1 and CRFB5, and CRFB2 and CRFB5, have been identified in fish, but the studies about preferential receptor usage of different fish IFN subtypes are rather limited. In this study, the three receptor chains of type I IFNs named as On-CRFB1, On-CRFB2 and On-CRFB5 were identified in Nile tilapia, Oreochromis niloticus. These three genes were constitutively expressed in all tissues examined, with the highest expression level observed in muscle and liver, and were rapidly induced in liver following the stimulation of poly(I:C). Interestingly, it is possible that all three subtypes of tilapia IFNs are able to signal through two pairs of the receptor subunits, On-CRFB1 and On-CRFB5, and On-CRFB2 and On-CRFB5. More importantly, tilapia group I IFNs (On-IFNd and On-IFNh) preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5, and group II IFNs (On-IFNc) preferentially signal through a receptor complex comprised of On-CRFB2 and On-CRFB5. The present study thus provides new insights into the receptor usage of group I and group II IFNs in fish.

Unique duplication of IFNh genes in Nile tilapia (Oreochromis niloticus) reveals lineage-specific evolution of IFNh in perciform fishes

Fish appear to harbour a complex type I IFN repertoire containing subgroups a, b, c, d, e, f, and h, and IFNh is only reported in perciform fishes. However, no multiple copies of IFNh gene has been identified in fish to date. In this study, two IFNh genes named On-IFNh1 and On-IFNh2 were cloned from Nile tilapia, Oreochromis niloticus. The predicted proteins of On-IFNh1 and On-IFNh2 contain several structural features known in type I IFNs, and estimation of divergence time revealed that these two genes may have arisen from a much recent local duplication event. On-IFNh genes were constitutively expressed in all tissues examined, with the highest expression level observed in gill, and were rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, both recombinant On-IFNh1 and On-IFNh2 trigger a relative delayed but sustained induction of interferon-stimulated genes (ISGs), whereas recombinant On-IFNc elicits a rapid and transient expression of ISGs in vivo. The present study thus contributes to a better understanding of the functional properties of tilapia interferons, and also provides a new insight into the evolution of IFNh in fish.

Exposure to low temperatures suppresses the production of B-cell activating factor via TLR3 in BEAS-2B cells

Acute viral respiratory tract infections (RTIs) are commonly associated with cold weather; however, the mechanism behind this is still unclear. Secretory IgA (sIgA) mainly contributes to the immune response against pathogenic microorganisms in the respiratory tract. Certain pathogen-associated molecular patterns (PAMPs) induce the expression of B-cell activating factor (BAFF) in epithelial cells, macrophages, and dendritic cells. BAFF transforms B cells into plasma cells, which leads to the mass production of immunoglobulins, including IgA, on the mucosal epithelium. However, no studies have described the relationship between cold exposure and BAFF and/or sIgA in RTI. The aim of our study was to determine this relationship in vitro by investigating the effect of low temperature on BAFF production by BEAS-2B cells after the addition of toll-like receptor (TLR) ligands. We showed stimulation of polyinosinic:polycytidylic acid (poly I:C), which led BEAS-2B to produce interferon (IFN)-β. IFN-β itself induced BEAS-2B cells to produce BAFF. Janus kinase inhibitor I decreased the amount of BAFF produced in BEAS-2B cells upon stimulation with IFN-β and poly I:C. Significantly less BAFF was produced post-poly I:C stimulation in low-temperature conditions than in normal-temperature conditions (mean ± SD: 41.2 ± 23.3 [33 °C] vs. 138.3 ± 7.1 pg/mL [37 °C], P = 0.05). However, the low-temperature condition itself was not cytotoxic. The stimulation of poly I:C produced BAFF from BEAS2B cells via IFN-β production and the JAK/signal transducer and activator of transcription pathway played an important role in BAFF production in BEAS-2B cells. Cold exposure reduced BAFF production by BEAS2B cells after stimulation with the TLR3 ligand. Cold exposure may, therefore, suppress the production of BAFF, resulting in the inhibition of IgA secretion in the bronchial epithelium, which explains the increased frequency of RTIs in cold weather.

Functional characterization of a group II interferon, IFNc in the perciform fish, Nile tilapia (Oreochromis niloticus)

Interferons are a family of class II α-helical cytokines playing vital roles in antiviral immune response, and little information is available to date regarding the interferon system of tilapia. In this study, a type I IFN gene, named On-IFNc, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-IFNc contains several structural features known in type I IFNs, and On-IFNc was clustered together with the known IFNc in fish into a separated clade in the phylogenetic tree. On-IFNc gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, recombinant On-IFNc has been proven to markedly induce the expression of the antiviral effectors, Mx and viperin, the signalling components, STAT1, STAT2, and IRF9, and the transcription factors, IRF3 and IRF7, as well as the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. Furthermore, recombinant On-IFNc has been proven to possess antiviral activity against ISKNV. The present study thus contributes to a better understanding of the functional properties of the type I IFN system in tilapia.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells

ObjectiveThe importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells.Materials and methodsCellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA.ResultsLL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function.ConclusionsLL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Transcriptional regulation of chicken leukocyte cell-derived chemotaxin 2 in response to toll-like receptor 3 stimulation.

ObjectiveLeukocyte cell-derived chemotaxin 2 (LECT2) is associated with several physiological processes including inflammation, tumorigenesis, and natural killer T cell generation. Chicken LECT2 (chLECT2) gene was originally identified as one of the differentially expressed genes in chicken kidney tissue, where the chickens were fed with different calcium doses. In this study, the molecular characteristics and gene expression of chLECT2 were analyzed under the stimulation of toll-like receptor 3 (TLR3) ligand to understand the involvement of chLECT2 expression in chicken metabolic disorders.MethodsAmino acid sequence of LECT2 proteins from various species including fowl, fish, and mammal were retrieved from the Ensembl database and subjected to Insilco analyses. In addition, the time- and dose-dependent expression of chLECT2 was examined in DF-1 cells which were stimulated with polyinosinic:polycytidylic acid (poly [I:C]), a TLR3 ligand. Further, to explore the transcription factors required for the transcription of chLECT2, DF-1 cells were treated with poly (I:C) in the presence or absence of the nuclear factor κB (NFκB) and activated protein 1 (AP-1) inhibitors.ResultsThe amino acid sequence prediction of chLECT2 protein revealed that along with duck LECT2 (duLECT2), it has unique signal peptide different from other vertebrate orthologs, and only chLECT2 and duLECT2 have an additional 157 and 161 amino acids on their carboxyl terminus, respectively. Phylogenetic analysis suggested that chLECT2 is evolved from a common ancestor along with the actinopterygii hence, more closely related than to the mammals. Our quantitative polymerase chain reaction results showed that, the expression of chLECT2 was up-regulated significantly in DF-1 cells under the stimulation of poly (I:C) (p<0.05). However, in the presence of NFκB or AP-1 inhibitors, the expression of chLECT2 is suppressed suggesting that both NFκB and AP-1 transcription factors are required for the induction of chLECT2 expression.ConclusionThe present results suggest that chLECT2 gene might be a target gene of TLR3 signaling. For the future, the expression pattern or molecular mechanism of chLECT2 under stimulation of other innate immune receptors shall be studied. The protein function of chLECT2 will be more clearly understood if further investigation about the mechanism of LECT2 in TLR pathways is conducted.

Grass carp (Ctenopharyngodon idella) RSK2 protects cells anti-apoptosis by up-regulating BCL-2

In mammals, toll-like receptor 3 (TLR3) is capable of recognizing double-stranded RNA and then initiates transcription of IFN-β. TLR3 can activate the innate immune system by phosphorylating extracellular signal-regulated kinase 1 (ERK1) in the mitogen-activated protein kinase (MAPK) pathway. As a downstream signaling protein of ERK1, ribosomal protein S6 kinase alpha 3 (RSK2) is activated through the “classical” MAPK pathway. So RSK2 plays a critical role in response to innate immune system induced by TRL3. However, the innate immune mechanism of RSK2 remains indistinct in fish. In this study, we cloned and characterized a full length cDNA sequence of RSK2 from Ctenopharyngodon idella (named CiRSK2, MH844551). The full length cDNA of CiRSK2 is 3930 bp with a coding sequence of 2202 bp encoding a polypeptide of 734 amino acids. The expression of CiRSK2 was ubiquitous and significantly up-regulated under the stimulation of poly (I:C) in eight different tissues of C. idella and C. idella kidney cells (CIK). In addition, poly (I:C) stimulation also up-regulated the expression of CiERK1 mRNA in CIK cells and the phosphorylation of CiERK1. We also demonstrated that the activated CiERK1 interacted with CiRSK2 by CO-IP assay and immunofluorescence assay. To further investigate the relationship between CiRSK2 and CiERK1, we performed subcellular localization of CiRSK2 at different periods of CiERK1 stimulation. The result showed that CiERK1 can make CiRSK2 enter the nucleus. Subsequently, we found that CiRSK2 increased the transcriptional level of CiBCL-2 and protein level of CiBCL-2 significantly. Then cell apoptosis was inhibited to a certain extent. Overall, our results suggested that CiRSK2 plays important roles in fish innate immunity and is able to inhibit cell apoptosis by up-regulating CiBCL-2.

Molecular Characterization and Expression Analysis of Clathrin-Associated Adaptor Protein 3-δ Subunit 2 (AP3S2) in Chicken

A chicken clathrin-associated adaptor protein 3-δ subunit 2 (AP3S2) is a subunit of AP3, which is involved in cargo protein trafficking to target membrane with clathrin-coated vesicles. AP3S2 may play a role in virus entry into host cells through clathrin-dependent endocytosis. AP3S2 is also known to participate in metabolic disease developments of progressions, such as liver fibrosis with hepatitis C virus infection and type 2 diabetes mellitus. Chicken <italic>AP3S2</italic> (<italic>chAP3S2</italic>) gene was originally identified as one of the differentially expressed genes (DEGs) in chicken kidney which was fed with different calcium doses. This study aims to characterize the molecular characteristics, gene expression patterns, and transcriptional regulation of <italic>chAP3S2</italic> in response to the stimulation of Toll-like receptor 3 (TLR3) to understand the involvement of chAP3S2 in metabolic disease in chicken. As a result, the structure prediction of <italic>chAP3S2</italic> gene revealed that the gene is highly conserved among AP3S2 orthologs from other species. Evolutionarily, it was suggested that chAP3S2 is relatively closely related to zebrafish, and fairly far from mammal AP3S2. The transcriptional profile revealed that <italic>chAP3S2</italic> gene was highly expressed in chicken lung and spleen tissues, and under the stimulation of poly (I:C), the <italic>chAP3S2</italic> expression was down-regulated in DF-1 cells (<italic>P</italic>&lt;0.05). However, the presence of the transcriptional inhibitors, BAY 11-7085 (Bay) as an inhibitor for nuclear factor κB (NFκB) or Tanshinone IIA (Tan-II) as an inhibitor for activated protein 1 (AP-1), did not affect the expressional level of <italic>chAP3S2</italic>, suggesting that these transcription factors might be dispensable for TLR3 mediated repression. These results suggest that <italic>chAP3S2</italic> gene may play a significant role against viral infection and be involved in TLR3 signaling pathway. Further study about the transcriptional regulation of <italic>chAP3S2</italic> in TLR3 pathways and the mechanism of chAP3S2 upon virus entry shall be needed.

Toll-like receptor signaling induces the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes

BackgroundLympho-epithelial Kazal-type inhibitor (LEKTI) tightly controls the activities of serine proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 in the epidermis. LEKTI is known to be an essential molecule for the epidermal skin barrier, as demonstrated by SPINK5 nonsense mutation, which results in Netherton syndrome. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and produce inflammatory cytokines, chemokines, and antimicrobial peptides. However, the effect of TLR signaling on the expression of LEKTI is not clear. ObjectiveTo investigate whether TLR signaling can affect expression of LEKTI in epidermal keratinocytes. MethodsWe stimulated a panel of TLR ligands and investigated the expression of LEKTI in normal human epidermal keratinocytes (NHEKs). We further measured trypsin or chymotrypsin-like serine protease activity in NHEK cultured media under stimulation with TLR3 ligand, poly (I:C). Immunostaining for LEKTI was performed using skin samples from skin infectious diseases. ResultsTLR1/2, 3, 5, and 2/6 ligands induced the expression of LEKTI in NHEKs. The trypsin or chymotrypsin-like serine protease activity in NHEKs was up-regulated with the stimulation of poly (I:C). The gene expressions of KLK6, KLK10, KLK11, and KLK13 were also increased by poly (I:C). An immunohistochemical analysis demonstrated that the expression of LEKTI was up-regulated in the lesions of varicella, pyoderma, and rosacea. ConclusionsTLR signaling induces the expression of LEKTI in epidermal keratinocytes, which might contribute to the control of aberrant serine protease activities in inflammatory skin diseases.

Grass carp (Ctenopharyngodon idella) STAT3 regulates the eIF2α phosphorylation through interaction with PKR

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an important role in growth, multiplication, differentiation and participates in inflammation, tumorigenesis, metabolic disorders and immune response. STAT3 is a protein that shuttles between the nucleus and cytoplasm. Compared to the STAT3 in cell nucleus, we did not know the function of STAT3 in cytoplasm for a long time. Some recent studies have shown that cytoplasmic STAT3 regulates autophagy through the interaction with the double-stranded RNA-activated protein kinase (PKR), which plays an important role in cellular antiviral response. Fish is a good target for developmental and comparative immunology. In the present study, we found that the expression of grass carp (Ctenopharyngodon idella) STAT3 (CiSTAT3) was ubiquitous and significantly up-regulated under the stimulation of poly I:C. To explore the potential function of fish cytoplasmic STAT3 in the antiviral signaling pathways, in this paper we analyzed the relationship between cytoplasmic CiSTAT3 and CiPKR. We demonstrated that the CiSTAT3 can combine with CiPKR in vivo and in vitro. The SH2 domain of CiSTAT3 and the C-terminus of CiPKR play an important role in this process. Moreover, the dimer of CiSTAT3 and CiPKR was formed under normal circumstances, however, it was dissociated under the induction of poly I:C. So, we guessed the binding of CiSTAT3 and CiPKR may regulate cell viability. It has also been shown that overexpression of CiSTAT3 in CIK cells can significantly reduce the level of p-eIF2α. On the contrary, the siRNA-mediated knockdown of CiSTAT3 and Stattic induction in CIK cells can up-regulate the p-eIF2α level. To further understand the relationship between CiSTAT3 and p-eIF2α level, we carried out the CiPKR-knockdown experiment. The result indicated that CiSTAT3 regulated the level of p-eIF2α through binding to CiPKR. In addition, overexpression of CiSTAT3 in CIK cells was able to improve the cell viability. These results above unraveled the molecular mechanism of fish cytoplasmic STAT3 regulating the eIF2α phosphorylation and cell viability. Therefore, the function of fish cytoplasmic STAT3 is similar to those of mammals.