Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA-146 function in the innate immune response of zebrafish embryos to Salmonella typhimurium infection [RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Danio rerio

SUBMITTER: Annemarie Meijer

PROVIDER: E-GEOD-45209 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88-dependent genes. This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents (allele hu3568, van der Vaart et al., 2013, Disease models & mechanisms 6, 841-854) or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection (4 dpi). The following treatment groups were used: homozygous mutants mock-injected with PBS/2%PVP 4 dpi, (2) wildtype siblings mock-injected with PBS/2%PVP 4dpi, (3) M. marinum-infected homozygous mutants 4dpi, (4) M. marinum-infected wildtype siblings 4dpi. 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 Mycobacterium marinum Mma20 bacteria into the caudal vein, or were mock-injected with buffer (PBS/2%PVP) as a control. After injections embryos were transferred into fresh egg water and incubated for 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for RNAseq analysis.

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MicroRNA-146 function in the innate immune response of zebrafish embryos to Salmonella typhimurium infection [RNA-seq]

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