MicroRNA gga-miR-200a-3p modulates immune response via MAPK signaling pathway in chicken afflicted with necrotic enteritis

International audienceAbstractMicroRNAs (miRNAs) are small non-coding RNAs that contribute to host immune response as post-transcriptional regulation. The current study investigated the biological role of the chicken (Gallus gallus) microRNA-200a-3p (gga-miR-200a-3p), using 2 necrotic enteritis (NE) afflicted genetically disparate chicken lines, 6.3 and 7.2, as well as the mechanisms underlying the fundamental signaling pathways in chicken. The expression of gga-miR-200a-3p in the intestinal mucosal layer of NE-induced chickens, was found to be upregulated during NE infection in the disease-susceptible chicken line 7.2. To validate the target genes, we performed an overexpression analysis of gga-miR-200a-3p using chemically synthesized oligonucleotides identical to gga-miR-200a-3p, reporter gene analysis including luciferase reporter assay, and a dual fluorescence reporter assay in cultured HD11 chicken macrophage cell lines. Gga-miR-200a-3p was observed to be a direct transcriptional repressor of ZAK, MAP2K4, and TGFβ2 that are involved in mitogen-activated protein kinase (MAPK) pathway by targeting the 3′-UTR of their transcripts. Besides, gga-miR-200a-3p may indirectly affect the expression of protein kinases including p38 and ERK1/2 at both transcriptional and translational levels, suggesting that this miRNA may function as an important regulator of the MAPK signaling pathway. Proinflammatory cytokines consisting of IL-1β, IFN-γ, IL-12p40, IL-17A, and LITAF belonging to Th1 and Th17-type cytokines, were upregulated upon gga-miR-200a-3p overexpression. These findings have enhanced our knowledge of the immune function of gga-miR-200a-3p mediating the chicken immune response via regulation of the MAPK signaling pathway and indicate that this miRNA may serve as an important biomarker of diseases in domestic animals

MicroRNA expression profiling in the lungs of genetically different Ri chicken lines against the highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) virus triggers infectious diseases, resulting in pulmonary damage and high mortality in domestic poultry worldwide. This study aimed to analyze miRNA expression profiles after infection with the HPAI H5N1 virus in resistant and susceptible lines of Ri chickens.For this purpose, resistant and susceptible lines of Vietnamese Ri chicken were used based on the A/G allele of Mx and BF2 genes. These genes are responsible for innate antiviral activity and were selected to determine differentially expressed (DE) miRNAs in HPAI-infected chicken lines using small RNA sequencing. A total of 44 miRNAs were DE after 3 days of infection with the H5N1 virus. Computational program analysis indicated the candidate target genes for DE miRNAs to possess significant functions related to cytokines, chemokines, MAPK signaling pathway, ErBb signaling pathway, and Wnt signaling pathway. Several DE miRNA-mRNA matches were suggested to play crucial roles in mediating immune functions against viral evasion. These results revealed the potential regulatory roles of miRNAs in the immune response of the two Ri chicken lines against HPAI H5N1 virus infection in the lungs

Chicken novel leukocyte immunoglobulin-like receptor subfamilies B1 and B3 are transcriptional regulators of major histocompatibility complex class I genes and signaling pathways

Objective The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. Methods Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. Results Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%–99%, while homologies between chicken and mammal proteins ranged between 13%–19%, and 13%–69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and β2-microglobulin and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. Conclusion These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, β2-microglobulin, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14

Objective This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. Results Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death

Analysis of Differentially Expressed Genes in Necrotic Enteritis-infected Fayoumi Chickens using RNA Sequencing

We identified and evaluated differentially expressed genes (DEGs) by RNA-Sequencing (RNA-Seq) in the intestinal mucosa of two Fayoumi chicken lines, M5.1 and M15.2, that are affected by necrotic enteritis (NE); these chicken lines share the same genetic background but have different major histocompatibility complexes. RNA-Seq generated over 49 and 40 million reads for lines M5.1 and M15.2, respectively. The alignment of these sequences with the Gallus gallus genome database revealed the expression of more than 14,500 genes in two lines, among which 581, 1270, and 1140 DEGs were detected when lines M15.2 and M5.1 were compared with the control and compared between each other. The analysis of all DEGs using the gene ontology database revealed annotations for 111 biological processes, 32 cellular components, and 17 molecular functions, and KEEG pathway mapping indicated that the DEGs were primarily involved in immunity, responses to various stimuli, and signal transduction. In addition, we analyzed 183 innate immune genes that were differentially expressed in NE-induced chicken lines, including 46 CD molecular genes, 89 immune-related genes, and 13 β-defensin genes with 3 lineage-specific duplications. Taken together, the transcriptional profiles showed that line M5.1 was more resistant to NE than line M15.2 and that differential gene expression patterns were associated with host genetic differences in resistance to NE. qRT-PCR and RNA-Seq analyses showed that all the genes examined had similar responses to NE (correlation coefficient R＝0.84 to 0.88, p<0.01) in both lines. To the best of our knowledge, this is the first study that describes NE-induced DEGs using RNA-seq in two lines with different levels of susceptibility to NE. These results will lead to increased insights on NE disease resistance mechanisms and the role of host genes in the control of the host immune response

Dataset on characterization of recombinant interleukin-23α, IL-12p40 and IL-23 complex protein, which activates JAK-STAT signaling pathway in chicken cell lines using immunocytochemical staining

The data herein is related to the research article entitled “Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex” [1] where we demonstrated that the chicken interleukin (IL)-23α, IL-12p40, and IL-23 complex regulates Th1, Th17, and Treg cytokine production through heterodimer receptors as well as a homodimer receptor consisting of IL-12Rβ1 and IL-23R, and activates the JAK/STAT signaling pathways. Here, we evaluated the effects of the recombinant chicken IL-23α, IL-12p40, and IL-23 complex protein on cell proliferation and nitric oxide (NO) production in chicken macrophage (HD11) and CU91 T cell lines. In addition, the expression of IL-6, IL-17A, and interferon-γ mRNA were upregulated in vivo and in vitro. Moreover, treatment with the chicken IL-23α, IL-12p40, and IL-23 complex activated phosphorylation of tyrosine and serine residues in JAK2, STAT1, TYK2, and SOCS1 in chicken cell lines. Keywords: Chicken, Interleukin-12, Proliferation, Nitric oxide, Signaling pathwa

Interleukin-34 Regulates Th1 and Th17 Cytokine Production by Activating Multiple Signaling Pathways through CSF-1R in Chicken Cell Lines

Interleukin-34 (IL-34) is a newly recognized cytokine with functions similar to macrophage colony-stimulating factor 1. It is expressed in macrophages and fibroblasts, where it induces cytokine production; however, the mechanism of chicken IL-34 (chIL-34) signaling has not been identified to date. The aim of this study was to analyze the signal transduction pathways and specific biological functions associated with chIL-34 in chicken macrophage (HD11) and fibroblast (OU2) cell lines. We found that IL-34 is a functional ligand for the colony-stimulating factor receptor (CSF-1R) in chicken cell lines. Treatment with chIL-34 increased the expression of Th1 and Th17 cytokines through phosphorylation of tyrosine and serine residues in Janus kinase (JAK) 2, tyrosine kinase 2 (TYK2), signal transducer and activator of transcription (STAT) 1, STAT3, and Src homology 2-containing tyrosine phosphatase 2 (SHP-2), which also led to phosphorylation of NF-κB1, p-mitogen-activated protein kinase kinase kinase 7 (TAK1), MyD88, suppressor of cytokine signaling 1 (SOCS1), and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Taken together, these results suggest that chIL-34 functions by binding to CSF-1R and activating the JAK/STAT, nuclear factor κ B (NF-κB), and mitogen-activated protein kinase signaling pathways; these signaling events regulate cytokine expression and suggest roles for chIL-34 in innate and adaptive immunity

Guided Wrinkling of Hierarchically Structured Nanoporous Gold Films for Improved Surface‐Enhanced Raman Scattering Performance

Abstract Plasmonic nanostructured metals have many advantages for applications in high‐performance surface‐enhanced Raman scattering (SERS) spectroscopy. In particular, unique designing nanostructures with bicontinuous ligaments surrounded by cylindrical voids with tunable dense pores from a few to hundreds of nanometers can be utilized for the high‐performance SERS‐active substrate. Here, a fabrication strategy is reported to prepare hierarchically arranged micro/nanostructures of wrinkled nanoporous gold (WNPG) films, which involves laminating of the dealloyed Au film on the heat‐shrinkable shape‐memory polymer film and geometrical modulation of the substrate. As a result, the various types of WNPG films are crafted with a remarkable density of cracks in the structured surface area. Specifically, the WNPG films consisting of multilayered overlapping features are explored and used as the SERS‐active substrate. This dual porosity coupled with localized surface plasmon resonance estimated by numerical simulation in a suitable model of bicontinuous ligaments is found to be the core mechanism for the enhancement of SERS sensitivity, which quantitatively characterizes the “hot spots” from the surface to interlayers. These suggested characteristic features are fully assessed by applying a series of dye molecules and DNA strands on the prepared SERS substrate, demonstrating the enhanced intensity of the Raman scattering signals on the optimized WNPG surface

Yeast-Based Genetic Interaction Analysis of Human Kinome

Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human–yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role