Transcriptomic analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium Marinum. Danio rerio

The mechanisms that govern the neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilize an innovative binary approach in zebrafish to perform in depth profiling of the initial response of neutrophils to Mycobacterium marinum, by in vivo biotinylation of neutrophil nuclei and subsequent rapid isolation directly from the in vivo context. RNA-seq and differential expression analysis of the neutrophil nuclear transcriptome reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, wu:fb15h11 (NLRC3 ortholog) and il1β. Crispr/Cas9-mediated knockdown of caspase b, which mediates its effects by the proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterium infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial response to mycobacterial infection in neutrophils. Further perturbation of the IEGs revealed egr3 as a key transcription factor regulating il1β transcription. Overall design: We report the design of a binary approach in zebrafish that allows for profiling of the initial response of neutrophils to Mycobacterium Marinum by in vivo biotinylation of neutrophil nuclei and subsequent rapid isolation directly from the in vivo context allowing for RNA-seq analysis of the active transcriptome.