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Title: Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection      
Transcriptome or Gene expression
Pathogenic mycobacteria have the ability to survive within macrophages and persist inside granulomas composed of host immune cells. The complex host-pathogen interactions that determine the outcome of a mycobacterial infection process result in marked alterations of the host gene expression profile. Here we used the zebrafish model to investigate the specificity of the host response to infections with two mycobacterium strains that give distinct disease outcomes: an acute disease with early lethality or a chronic disease with granuloma formation, caused by Mycobacterium marinum strains Mma 20 and E11, respectively. We performed a microarray study of different stages of disease progression in adult zebrafish and found that the acute and the chronic strains evoked partially overlapping host transcriptome signatures, despite that they induce profoundly different disease phenotypes. Both strains affected many signaling cascades, including Wnt and Tlr pathways. Interestingly, the strongest differences were observed at the initial stage of the disease. The immediate response to the acute strain was characterized by higher expression of genes encoding MHC class I proteins, matrix metalloproteinases, transcription factors, cytokines and other common immune response proteins. In contrast, small GTPase and histone gene groups showed higher expression in response to the chronic strain. We also found that nearly 1,000 mycobacterium-responsive genes overlapped between the expression signatures of infected zebrafish adults and embryos at different stages of granuloma formation. Since adult zebrafish possess an adaptive immune system similar to mammals and zebrafish embryos rely solely on innate immunity, this overlap indicates a major contribution of the innate component of the immune system in the response to mycobacterium infection. Taken together, our comparison of the transcriptome responses involved in acute versus chronic infections and in the embryonic versus adult situation provides important new leads for investigating the mechanism of mycobacterial pathogenesis. Overall design: Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols ( Infection experiments were approved by the local animal welfare committee (DEC) of the VU University medical center and of Leiden Univeristy. Infection experiments with adult fish were performed on young males selected from a wild type laboratory-breeding colony and acclimated to their new environment for one week in a quarantine area. These fish were kept at 28˚C on a 12:12 h light/dark rhythm throughout the experiment. Groups of 30 fish, infected with the same dose and strain of mycobacteria, were kept in small fish tanks (10 l) with their own separate filtering system (Eheim Ecco). Zebrafish were inoculated intraperitoneally as previously described (Meijer et al., 2004) with approximately 10000 bacteria or with phosphate-buffered saline (PBS) as a control. For the acute infection study with E11 and Mma20 strains, 3 fish per group were sacrificed at 1 and 6 days post infection (dpi) and used for microarray analysis. For comparison with the end stage of chronic E11 infection we used RNA samples from our previously published chronic infection study (Meijer et al., 2005; control fish c2 and infected fish i2) and additional RNA samples (2 controls, 2 infected) from a similar infection experiment. All chronically infected fish showed overt signs of fish tuberculosis, including lethargy and skin ulcers. Histological examination of fish from the same experiments confirmed that the pathology of infected fish corresponded to fish tuberculosis (Van der Sar et al., 2004) and that no characteristics of the disease were present in the control fish. Infection experiments at the embryonic stage were performed using mixed egg clutches from different pairs of AB strain zebrafish. Embryos were grown at 28,5 -30 °C in egg water (60µg/ml Instant Ocean see salts) and for the duration of bacterial injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). Embryos were staged at 28 hours post fertilization (hpf) by morphological critera (Kimmel et al.) and approximately 50 cfu of E11 bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Infection experiments were carried out in triplicate on separate days and pools of 15-20 embryos were taken at 2, 24 and 120 hours post infection (hpi).
Danio rerio
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