An alternative receptor to poly I:C on cell surfaces for interferon induction

Not‐self or denatured nucleic acids are recognized by pattern recognition receptors localized mainly in endosomes and cytoplasm, such as Toll‐like receptor (TLR) 3, TLR7, TLR9, retinoic acid‐inducible gene‐I, DNA‐dependent activator of IFN‐regulatory factors and other receptors. The binding of polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that robustly induces type I interferon, to a putative cell‐surface receptor on a rabbit kidney cell line, RK13, has been analyzed by the authors and RK13 cells found to capture poly I:C in a specific fashion with sufficient affinity. These findings suggest that an alternative receptor to poly I:C participates in the induction of type 1 interferon, which localizes on cell surfaces. Although the nature of this molecule has not yet been identified, accumulating evidence has led the present authors to speculate that there are undefined classes of RNA‐recognition molecules on cell surfaces and that these are unlikely to be categorized as previously reported dsRNA receptors. Although many years have passed since this possibility was first reported by the present authors, it remains attractive. In this article, previously reported cell‐surface dsRNA receptors are reviewed in comparison with other receptors reported to date that are firmly involved in the innate immune‐sensing of nucleic acids.     

聚肌胞免疫刺激下仔猪外周血单核细胞的基因表达分析

聚肌胞(Poly I:C)是一种天然双链RNA(Double strand RNA, dsRNA)的拟似物,能够模拟病毒感染后所形成的dsRNA及刺激机体产生抗病毒免疫反应。文章以抗病力存在差异的大蒲莲和长白仔猪为研究对象,分离外周血单核细胞(Peripheral blood mononuclear cell, PBMC),在20 μg/mL的Poly I:C的免疫刺激下体外培养24 h,对影响免疫应答过程中的7个细胞因子(IRF3、IL6、IL8、IL10、TNFα、IFNγ和IFNα)和3个模式识别受体(TLR3、TLR4和RIG1)利用实时荧光定量PCR检测Poly I:C免疫刺激组相对于对照组的基因表达变化倍数。结果表明：检测的大部分细胞因子和受体(6个)表达量变化倍数很大,其中3种白细胞介素IL6、IL8和IL10免疫刺激变化倍数最大,平均变化倍数分别为20.71、10.87和5.18倍。对不同个体和品种间的比较发现,不仅大蒲莲和长白两品种间(大蒲莲猪的变化倍数平均高于长白猪)而且同品种的3头全同胞仔猪间对Poly I:C免疫刺激的应答也存在较大的变化。文章利用Poly I:C体外模拟dsRNA对PBMC的感染,为下一步筛选仔猪对Poly I:C刺激的免疫应答基因及鉴定大蒲莲猪特殊的抗性基因奠定了基础。     

Characterization of miRNAs involved in response to poly(I:C) in porcine airway epithelial cells

MicroRNAs (miRNA) have been implicated in a variety of pathological conditions including infectious diseases. Knowledge of the miRNAs affected by poly(I:C), a synthetic analog of viral double‐stranded RNA, in porcine airway epithelial cells (PAECs) contributes to understanding the mechanisms of swine viral respiratory diseases, which bring enormous economic loss worldwide every year. In this study, we used high throughput sequencing to profile miRNA expression in PAECs treated with poly(I:C) as compared to the untreated control. This approach revealed 23 differentially expressed miRNAs (DEMs), five of which have not been implicated in viral infection before. Nineteen of the 23 miRNAs were down‐regulated including members of the miR‐17‐92 cluster, a well‐known polycistronic oncomir and extensively involved in viral infection in humans. Target genes of DEMs, predicted using bioinformatic methods and validated by luciferase reporter analysis on two representative DEMs, were significantly enriched in several pathways including transforming growth factor‐β signaling. A large quantity of sequence variations (isomiRs) were found including a substitution at position 5, which was verified to redirect miRNAs to a new spectrum of targets by luciferase reporter assay together with bioinformatics analysis. Twelve novel porcine miRNAs conserved in other species were identified by homology analysis together with cloning verification. Furthermore, the expression analysis revealed the potential importance of three novel miRNAs in porcine immune response to viruses. Overall, our data contribute to clarifying the mechanisms underlying the host immune response against respiratory viruses in pigs, and enriches the repertoire of porcine miRNAs.     

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C‐glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C‐glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C‐glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C‐glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C‐glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C‐glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over‐expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.     

TLR3 engagement induces IRF‐3‐dependent apoptosis in androgen‐sensitive prostate cancer cells and inhibits tumour growth in vivo

Toll‐like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double‐stranded RNA analogue poly I:C induces apoptosis of androgen‐sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3‐mediated apoptosis and the in vivo efficacy of poly I:C‐based therapy. We show that interferon regulatory factor‐3 (IRF‐3) signalling plays an essential role in TLR3‐mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE‐1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well‐established human androgen‐sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C‐treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF‐3 in both human normal and PCa clinical samples, potentially envisaging poly I:C‐based therapy for PCa.     

Novel antagonist antibody to TLR3 blocks poly(I:C)-induced inflammation in vivo and in vitro

Toll-like receptor 3 (TLR3) binds and signals in response to dsRNA and poly(I:C), a synthetic double stranded RNA analog. Activation of TLR3 triggers innate responses that may play a protective or detrimental role in viral infections or in immune-mediated inflammatory diseases through amplification of inflammation. Two monoclonal antibodies, CNTO4685 (rat anti-mouse TLR3) and CNTO5429 (CDRs from CNTO4685 grafted onto a mouse IgG1 scaffold) were generated and characterized. These mAbs bind the extracellular domain of mouse TLR3, inhibit poly(I:C)-induced activation of HEK293T cells transfected with mTLR3, and reduce poly(I:C)-induced production of CCL2 and CXCL10 by primary mouse embryonic fibroblasts. CNTO5429 decreased serum IL-6 and TNFα levels post-intraperitoneal poly(I:C) administration, demonstrating in vivo activity. In summary, specific anti-mTLR3 mAbs have been generated to assess TLR3 antagonism in mouse models of inflammation.     

RNA‐sequencing profiles hippocampal gene expression in a validated model of cancer‐induced depression

To investigate the pathophysiology of cancer‐induced depression (CID), we have recently developed a validated CID mouse model. Given that the efficacy of antidepressants in cancer patients is controversial, it remains unclear whether CID is a biologically distinct form of depression. We used RNA‐sequencing (RNA‐seq) to investigate differentially expressed genes (DEGs) in hippocampi of animals from our CID model relative a positive control model of depressive‐like behavior induced with chronic corticosterone (CORT). To validate RNA‐seq results, we performed quantitative real‐time RT‐PCR (qRT‐PCR) on a subset of DEGs. Enrichment analysis using DAVID was performed on DEGs to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP). qRT‐PCR results significantly predicted RNA‐seq results. RNA‐seq revealed that most DEGs identified in the CORT model overlapped with the CID model. Enrichment analyses identified KEGG pathways and GO:BP terms associated with ion homeostasis and neuronal communication for both the CORT and CID model. In addition, CID DEGs were enriched in pathways and terms relating to neuronal development, intracellular signaling, learning and memory. This study is the first to investigate CID at the mRNA level. We have shown that most hippocampal mRNA changes that are associated with a depressive‐like state are also associated with cancer. Several other changes occur at the mRNA level in cancer, suggesting that the CID model may represent a biologically distinct form of a depressive‐like state.     

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full‐length complementary (c)DNA of interleukin‐1 receptor‐associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine–threonine‐specific protein kinase catalytic domain and a C‐terminal domain. The amino‐acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84·5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up‐regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up‐regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.     

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini‐barcode

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century‐old museum specimens and shown to be useful as mini‐barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode.     

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3.