Abstract
The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription.
Highlights
In 2015 10.4 million people developed tuberculosis and 1.8 million people died from the disease[1]
We demonstrate that knockout of caspase b, which enhances processing and secretion of il1β, increases bacterial burden and prevents macrophage migration to sites of infection in a process that results in extracellular growth of the mycobacteria
Investigations focused on the early host response to mycobacteria are important, since both epidemiological and genetic studies suggest that the early phases of infection, before active or latent tuberculosis is established, might represent a therapeutic window where the mycobacterium can be eliminated[21]
Summary
In 2015 10.4 million people developed tuberculosis and 1.8 million people died from the disease[1]. In Mycobacterium tuberculosis (MTB), the innate immune phase is usually initiated by the phagocytosis of bacteria in the lung by macrophages, followed by the accumulation of macrophages, neutrophils, dendritic cells and innate lymphoid cells[2] As they are recruited, some cells get infected by the expanding population of mycobacterium and early granulomas start to form[2]. To identify neutrophil-specific effector mechanisms capable of controlling mycobacterial infection, we have developed a novel transgenic zebrafish model system based on the method developed by Trinh and Chong et al, adapted for the in vivo biotinylation and the subsequent isolation of the neutrophil nuclei[11] This model circumvents previous problems with the variability of short-lived neutrophils following isolation from blood by rapidly sequestering the nuclei. We show that egr[3], an early growth response transcription factor, plays a role in the modulation of transcription of il1β
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