MicroRNA-146 function in the innate immune response of zebrafish embryos to Salmonella typhimurium infection
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Annemarie Meijer
PROVIDER: E-GEOD-45410 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:This study reports on infection-inducible miRNAs in zebrafish. Using a custom-designed microarray platform for miRNA expression we found that miRNAs of the miR-21, miR-29, and miR-146 families were commonly induced by infection of zebrafish embryos with Salmonella typhimurium and by infection of adult fish with Mycobacterium marinum. A custom-designed Agilent zebrafish 8x15k miRNA platform was used to profile miRNA expression in zebrafish embryos infected with Salmonella typhimurium strain SL1027 and adult zebrafish infected with Mycobacterium marinum strain Mma20 . The 15k design contained a duplicate of 7604 probes of 60-oligonucleotide length. The probes consisted of 2x22 nucleotide sequences antisense to mature miRNAs separated by a spacer of 8 nucleotides (CGATCTTT) and with a second spacer with the same sequence at the end. From 7604 probes 546 were designed for left (5') and right (3') arms of the hairpins of zebrafish miRNAs known in miRBase, while the remainder 7058 probes corresponded to predicted hairpin structures in the zebrafish genome that might include additional miRNAs but were not considered in this study. Zebrafish embryos were infected at 28 hours post fertilization (hpf) by injecting 200-250 colony forming units of Salmonella typhimurium into the caudal vein and miRNA profiles of infected embryos were compared to control embryos injected with PBS (phosphate buffered saline) at 8 hours post-infection (hpi; 3 biological replicates and 2 technical replicates per each sample). Adult zebrafish were infected with 10000 colony forming units of Mycobacterium marinum and miRNA profiles were compared to PBS-injected control fish at 6 days post infection (dpi; 3 biological replicates). For dual color hybridization of the Agilent chips miRNA samples from infected zebrafish were labeled with Hy3 and samples from control fish were labeled with Hy5.
2013-09-16 | E-GEOD-45409 | biostudies-arrayexpress
Project description:We used zebrafish embryos as an in vivo system to investigate the role of the microRNA-146 family (consisting of 2 members miR-146a and miR-146b) in the innate immune response to S. typhimurium infection. To determine the role of miR-146 microRNAs in the response to S. typhimurium infection we used Illumina RNA sequencing to compare the mRNA expression profiles of control embryos versus embryos with knockdown of miR-146a and miR-146b. RNA sequencing analysis of miR-146 knockdown embryos showed no major effects on pro-inflammatory gene expression or on the expression of transcriptional regulators and signal transduction components of the immune response. In contrast, apoliprotein-mediated lipid transport emerged as an infection-inducible pathway under miR-146 knockdown conditions, suggesting a function of miR-146 in regulating lipid metabolism during inflammation. Embryos were injected at the one cell stage with a combination of two morpholinos targeting miR-146a and miR-146b, or with the standard control morpholino from GeneTools. Subsequently, at 28 hours post fertilzation (hpf) they were infected by injecting 200-250 colony forming units of S. typhimurium strain SL1027 into the caudal vein, or mock-injected with PBS. RNA was isolated at 8 hours post injection (hpi) for Illumina RNAseq analysis. Two independent experiments were performed for RNAseq analysis of biological duplicates.
2013-09-16 | E-GEOD-45209 | biostudies-arrayexpress
Project description:Deficiency in the protein-tyrosine phosphatase SHP1/PTPN6 is linked with hematological malignancies and chronic inflammatory diseases. Here we exploited the embryonic and larval stages of zebrafish as an animal model to study ptpn6 function in the sole context of innate immunity. We show that ptpn6 knockdown induces a spontaneous inflammation-associated phenotype at the late larval stage, which was microbe-independent and enhanced instead of suppressed by glucocorticoids. When challenged with Salmonella typhimurium or Mycobacterium marinum at earlier stages of development, the innate immune system was hyperactivated to a contra-productive level that impaired the control of these pathogenic bacteria. These results demonstrate the crucial regulatory function of ptpn6 in preventing host-detrimental effects of inflammation by imposing a tight control over the level of innate immune response activation. This microarray study was designed to determine the effect of morpholino knockdown of the ptpn6 gene on the innate immune response of zebrafish embryos during infection with Salmonella typhimurium. Three independent infection experiments were performed using mixed egg clutches of wild type zebrafish, which were a cross of the AB and TL strains. Each experiment consisted of the following four treatment groups: (1) uninfected control embryos, (2) S. typhimurium-infected control embryos, (3) uninfected ptpn6 knockdown embryos, and (4) S. typhimurium-infected ptpn6 knockdown embryos. Knockdown of ptpn6 was performed by micro-injecting embryos at the 1-2 cell stage with the ptpn6 morpholino, and control embryos were mock-injected with buffer. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200-250 colony forming units (CFU) of S. typhimurium SL1027 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h at 28M-BM-0C. After the incubation period, pools of 15-20 embryos per treatment group were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
2012-12-31 | E-GEOD-34930 | biostudies-arrayexpress
Project description:We use the zebrafish embryo model to study the similarities and differences in the innate immune response against three different bacterial pathogens. Therefore, we injected E. tarda, Salmonella, or M. marinum into the caudal vein of 28 hours post fertilization (hpf) zebrafish embryos and analysed their gene expression profile at 8 hours or 4 days after infection by microarrays. The results show that infections with the gram-negative bacteria E. tarda and S. typhimurium, which are lethal within 1-2 days, induce a strong early immune response at 8 hours after infection. In contrast, infection with M. marinum leads to a chronic infection that only induces a strong response at 4 days post infection. This microarray study was designed to determine the gene expression profile during infection with Salmonella typhimurium, Mycobacterium marinum, and Edwardsiella tarda. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Wildtypes injected with PBS 8 hours post infection (hpi), (2) S. typhimurium-infected wildtypes 8hpi, (3) wildtypes injected with PBS/2%PVP 8hpi , (4) M. marinum-infected wildtypes 8hpi, (5) wildtypes injected with PBS/2%PVP 4 days post infection (dpi), (6) M. marinum-infected wildtypes 4dpi, (7) wildtypes injected with PBS 8hpi (E. tarda control), (8) E. tarda-infected wildtypes 8hpi. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027, E. tarda or Mycobacteria marinum M20 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
2012-11-01 | E-GEOD-35474 | biostudies-arrayexpress
Project description:MyD88 is an adaptor protein in Toll-like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88, leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Salmonella typhimurium and Edwardsiella tarda to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. The data derived from these microarray experiments confirms the vital role of MyD88 in pathogen recognition and provides many leads for further research. This microarray study was designed to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Salmonella typhimurium and Edwardsiella tarda. Embryos used in this study are derived from an incross between parents heterozygous for the mutation. Both homozygous mutants and their wildtype siblings were selected by genotyping after being injected with the bacteria or PBS as control. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Homozygous mutants injected with PBS 8 hours post infection (hpi), (2) wildtype siblings injected with PBS 8hpi, (3) S. typhimurium-infected homozygous mutants 8hpi, (4) S. typhimurium-infected wildtype siblings 8hpi, (5) Homozygous mutants injected with PBS 8 hours post infection (hpi) (E.tarda control), (6) wildtype siblings injected with PBS 8hpi (E. tarda control), (7) E. tarda-infected homozygous mutants 8hpi, (8) E. tarda-infected wildtype siblings 8hpi. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027 or E. Tarda FL-F60, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
2012-12-10 | E-GEOD-39274 | biostudies-arrayexpress
Project description:Transcriptional profiling of the zebrafish embryonic host response to a systemic bacterial infection with Salmonella typhimurium (strain SL1027); comparison between traf6 knock-down and control morpholino treated embryos. All infection experiments were performed using mixed egg clutches of ABxTL strain zebrafish. Embryos injected with traf6 morpholino or a 5bp mismatch control morpholino were staged at 27 hours post fertilization (hpf) by morphological criteria and approximately 250 cfu of DsRed expressing Salmonella bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Pools of 20-40 infected and control embryos were collected 8 hours post infection (hpi). The whole procedure was preformed in triplicate on separate days. Total RNA of the biological triplicates was pooled using equal amounts of RNA prior to RNAseq library preparation.
2010-08-01 | E-GEOD-21024 | biostudies-arrayexpress
Project description:Transcriptional profiling of the zebrafish embryonic host response to a systemic bacterial infection with Salmonella typhimurium (strain SL1027); comparison between traf6 knock-down and control morpholino treated embryos. Two-way factorial dual colour design with factor 1 'infection with Salmonella enterica serovar Typhimurium (S. typhimurium)' and factor 2 'morpholino knock-down of traf6' using a common reference made from the sample pool. All infection experiments were performed using mixed egg clutches of ABxTL strain zebrafish. Embryos injected with traf6 morpholino or a 5bp mismatch control morpholino were staged at 27 hours post fertilization (hpf) by morphological criteria and approximately 250 cfu of DsRed expressing Salmonella bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Pools of 20-40 infected and control embryos were collected 8 hours post infection (hpi). For the microarray analysis, the whole procedure was preformed in triplicate on separate days. The triplicates are marked 1,2 and 3.
2010-08-01 | E-GEOD-20310 | biostudies-arrayexpress
Project description:We use the zebrafish embryo model to study the innate immune response against Staphylococcus epidermidis. Therefore, we injected S. epidermidis (and three controls groups) into the yolk at 2 hpf and samples at mutiple timepoints. Gene expression profiles were obtained at 6, 30, 54, 78, 102 and 126 hpi by microarrays. The results show that the gram-positive bacterium S. epidermidis induces a late immune response with a strong response at 102 hpi. This microarray study was designed to determine the gene expression profile during infection with Staphylococcus epidermidis. RNA was isolated from groups of embryos (20) at 6 timepoints during the infection. Wildtypes zebrafish embryos were micro-injected into the yolk (2hpf) with (1) 20 CFU of S. epidermdis O-47 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), (2) mock-injected with PVP as a control, (3) Needle insertion as control, (4) Non-injected as a control. After injections embryos were transferred into fresh egg water and incubated at 28M-BM-0C. At 8 hpf (6 h post infection), 32 hpf (30 h post infection), 56 hpf (54 h post infection), 80 hpf (78 h post infection), 104 hpf (102 h post infection) or 128 hpf (126 h post infection) twenty embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent. All treatment groups were analyzed using a common reference approach.
2013-04-01 | E-GEOD-37227 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-04-01 | E-GEOD-42847 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-06-01 | E-GEOD-49188 | biostudies-arrayexpress