查看更多>>摘要：MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPVencoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRTPCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKK beta), via binding to the target mRNA 3 & PRIME;-untranslated region, which fine-tuned the host NF -KB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.
查看更多>>摘要：Red palm weevil, Rhynchophorus ferrugineus, is an invasive and destructive pest that causes serious damages to palm trees. Like other invertebrates, red palm weevil relies solely on its innate immune response to fight invading microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming the red palm weevil larvae with heat-killed Bacillus thuringiensis specifically increased survival of the larvae during a secondary lethal infection with live bacteria, and B. thuringiensis primed larvae also showed a higher clearance efficiency for this bacterium, which indicated that the red palm weevil larvae possessed a strong immune priming response. The degree of enhanced immune protection was positively correlated with hemocyte proliferation and the level of phagocytic ability of hemocytes. Moreover, the red palm weevil larvae primed by B. thuringiensis induced the continuous synthesis of serotonin in the hemolymph, which in turn enhanced the phagocytic ability and pathogen clearance ability of the host, representing an important mechanism for the red palm weevil to achieve priming protection. Our findings reveal a specific immune priming of the red palm weevil larvae mediated by the continuous secretion of serotonin, and provide new insights into the mechanisms of in-vertebrates immune priming.
查看更多>>摘要：Gamma delta (gamma delta) T cells are highly enriched in mucosal barrier sites including intestinal tissues where microbial infections and tumors often originate in mammals. Human gamma delta 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 gamma delta 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 CD8 alpha molecules on circulating and lung gamma delta T cells. Moreover, poly(I:C) stimulation induced interferon (IFN)-gamma production from splenic and lung CD8 alpha+ gamma delta T cells while Cytosine-phosphate-Guanine oligodeoxynucleotides (CpG-ODN) 2007, a TLR21 ligand, stimulation induced IFN-gamma production by circulating gamma delta T cells. Neither poly(I:C) nor CpG-ODN 2007 stimulation elicited degranulation of gamma delta T cells. Additionally, the results revealed that CpG-ODN 2007 induced IFN-gamma production from TCR-stimulated gamma delta 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-gamma production or degranulation in gamma delta T cells. Taken together, a combination of CpG-ODN 2007 and anti-CD3e monoclonal antibodies (mAbs) can stimulate chicken gamma delta T cells and induce production of IFN-gamma by these cells while IFN-gamma production by gamma delta T cells induced by stimulation of poly(I:C) needs signals from other cells. These results suggest that chicken gamma delta T cells can sense invading pathogens via TLRs and produce IFN-gamma as a first line of defense.
查看更多>>摘要：In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the kappa light chain is replaced by lambda before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3' V and 5'J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5' IGLV and 3' IGLJ genes are used. A similar shift toward the use of more 5' IGKV and 3' IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most lambda(+) B cells are generated earlier, while kappa+ B cells develop from late precursors that already contain the lambda rearrangement. The initial lambda rearrangement is retained in both lambda(+) and kappa(+) B lymphocytes, and multiple lambda transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The kappa repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced lambda transcripts in kappa(+) B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.
查看更多>>摘要：The Nemo-like kinase (NLK) is an important serine/threonine-protein kinase in many signaling pathways. However, its function in crustaceans, such as shrimps, is still poorly understood and needs to be further explored. In the present study, the full-length cDNA of NLK from Litopenaeus vannamei (LvNLK) was cloned. The full-length LvNLK cDNA has 2497 bp, including an open reading frame (ORF) of 1524 bp encoding a protein with 507 amino acids and a predicted molecular mass of 56.1 kDa. Phylogenetic analysis revealed that LvNLK shared high similarities with NLK from other known species. Low-temperature stress markedly upregulated the expression of LvNLK. Its overexpression in hemocytes suppressed the expression of BCL2-associated X (Bax) and tumor protein P53 (p53) in vitro. Meanwhile, the BCL2 apoptosis regulator (Bcl-2), MDM2 proto-oncogene (MDM2), and Yin Yang 1 (YY1) were upregulated. Moreover, LvNLK silencing in vivo increased the susceptibility of shrimps to low temperature stress. The generation of ROS and the rate of hemocyte apoptosis also increased when LvNLK was silenced. Additionally, qPCR results indicated that LvNLK might participate in apoptosis via the p53 signaling pathway in vitro and in vivo. These results suggested that LvNLK is indispensable for the environmental adaptation of L. vannamei. Our current findings also demonstrated that NLK is evolutionarily conserved in crustaceans and provided insights into the environmental adaptation of invertebrates.
查看更多>>摘要：Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine, which plays a dual role in mammalian inflammation through both classical signaling (IL-6 binds to IL-6 receptor/IL-6R) and trans-signaling (IL-6 binds to soluble IL 6R). However, the function of IL-6, especially the regulatory mechanism of IL-6 trans-signaling in immunity and iron metabolism remains largely unclear in teleost. Here, L8824 cells (Ctenopharyngodon idella hepatic cells) were stimulated with blunt snout bream (Megalobrama amblycephala) IL-6 combination with sIL-6R protein (rmaIL6+rmasIL-6R/maIL-6 trans-signaling) or STAT3 inhibitor (c188-9), and RNA-sequencing, global transcriptional analyses. The enrichment analysis of GO and KEGG showed that maIL-6 trans-signaling is mainly involved in stress and inflammation response, and the activation of STAT3 is mainly related to cell proliferation, apoptosis and immune regulation. Furthermore, after treated L8824 cells with JAK2 inhibitors, it was found that the induction of IL-6 trans-signaling on the selected immune-related genes could be inhibited. These results implied that in early stage after rmaIL-6+rmasIL-6R treatment, the maIL-6 trans-signaling played an important role in the immune regulation through the JAK2/STAT3 pathway. By extending the rmaIL-6+rmasIL-6R treatment time, it was found that maIL-6 trans-signaling could promote the expression of iron metabolism related genes (ft, tf, tfr1, hamp and fpn1) in L8824 cells, indicating that maIL-6 trans-signaling may be involved in iron metabolism in the non-acute immune phase. Finally, after treated L8824 cells with JAK2 and STAT3 inhibitors, it was found that only tf and fpn1 were regulated by maIL-6 trans-signaling through the JAK2/STAT3 pathway. These findings provided novel insights into IL-6 trans-signaling regulatory mechanism in teleost, enriching our knowledge of fish immunity and iron metabolism.
查看更多>>摘要：Sebastes schlegelii (black rockfish) is a popular and economically important fish species in aquaculture. However, disease outbreaks have hindered the development of its cultivation. Antimicrobial peptides (AMPs) are a group of important components in fish innate immune system, that are active in the first line of defense against pathogens. The piscidin family, which are a group of fish-specific AMPs, have been isolated in a part of teleost but still poorly understood in S. schlegelii. In this study, three piscidin genes (Ss-piscidin1, 2, 3) are identified in S. schlegelii and their antibacterial activities and related mechanisms are analyzed. Three Ss-piscidins have conserved signal peptides but highly variable mature peptides and prodomains, and their mature regions all have predicted amphipathic and alpha-helical structures. Phylogenetic analysis shows that three Ss-piscidins cluster with different fish piscidin sequences into three sister clades, which correspond to three groups of fish piscidin family, respectively. Ss-piscidins have constitutive expressions in different tissues of healthy fish and enhanced expressions after Aeromonas salmonicida challenge. All three piscidins exhibit antibacterial activities, and are able to enhance bacterial membrane permeability and change bacterial morphology to different degrees, with a positive correlation observed among these activities. This suggests that three peptides exert their antibacterial activity through a "membrane-attack " mechanism. Moreover, hemolytic activities of three piscidins are also analyzed, and Ss-piscidin1, with low hemolytic ability and high antibacterial activity, is considered to be a possible candidate template for design of AMP drugs. Results in this study can promote a better understanding of immune responses in black rockfish and facilitate the future development of strategies in fish disease control in aquaculture.
查看更多>>摘要：Tumor necrosis factor-alpha (TNF-alpha) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-alpha is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-alpha (chTNF-alpha) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-alpha using a panel of newly developed anti-chTNF-alpha mouse monoclonal antibodies (mAbs). Using anti-chTNF-alpha mAbs, we determined the tissue expression of chTNF-alpha in lymphoid and non-lymphoid organs. A chTNF-alpha-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-alpha in the circulation of Clostridium perfringens , Eimeria , or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-alpha in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-alpha-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-alpha, and these anti-chTNF-alpha mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.
查看更多>>摘要：Integrins are transmembrane receptor heterodimers composed of alpha and beta subunits. They are known to mediate extracellular signals to promote cell adhesion and spreading, and are therefore essential for cellular immunity. However, proteins that bind to integrin cytoplasmic domains and mediate intracellular signaling to promote cell adhesion require identification. Calcium and integrin-binding protein 1 (CIB1) that binds to the integrin alpha-cytoplasmic domain has rarely been examined in insects. In this study, we found that 20-hydroxyecdysone promoted cell phagocytosis and spreading in Helicoverpa armigera. Transcriptomic analyses of hemocytes identified an integrin alpha gene (HaINT alpha-PS1) whose expression could be induced by either 20-hydroxyecdysone injection or bead challenge. Isothermal titration calorimetry assays showed that H. armigera CIB1-like (HaCIB1like) weakly bound to the cytoplasmic domain of HaINT alpha-PS1 in the presence of calcium. HaINT alpha-PS1 or HaCIB1-like knockdown inhibited hemocytic encapsulation and phagocytosis, and plasmatocyte spreading. Moreover, HaCIB1-like overexpression in a H. armigera epidermal cell line overexpanded cells and impaired cell phagocytosis. Thus, insect CIB1-like potentially interacted with integrin alpha-cytoplasmic domain and facilitated cell adhesion. This study enriches our understanding of the molecular mechanism underlying integrin-mediated cellular immunity in insects.
Jorgensen, Peter B.Eriksen, Lise L.Fenton, Thomas M.Bailey, Michael...
9页
查看更多>>摘要：Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood. Given the importance of pigs as models of human disease, as well as their role in livestock production, we adapted a recently developed protocol for the isolation of human GALT to assess the diversity, development and immune composition of large intestinal GALT in neonatal and adult pigs. We demonstrate that the large intestine of adult pigs contains two major GALT types; multifollicular submucosal GALT that we term submucosal lymphoid clusters (SLC) which develop prenatally, and as yet undescribed mucosal isolated lymphoid follicles (M-ILF), which arise after birth. Using confocal laser microscopy and flow cytometry, we additionally assess the microanatomy and lymphocyte composition of SLC and M-ILF, compare them to jejunal Peyer's patches (PP), and describe the maturation of these structures. Collectively, our results provide a deeper understanding of the diversity and development of GALT within the porcine large intestine.
NSTL
Elsevier