High content analysis of granuloma histology and neutrophilic inflammation in adult zebrafish infected with Mycobacterium marinum

Abstract

Infection of zebrafish with natural pathogen Mycobacterium marinum is a useful surrogate for studying the human granulomatous inflammatory response to infection by Mycobacterium tuberculosis. The adaptive immune system of the adult stage zebrafish offers an advance on the commonly used embryo infection model as adult zebrafish form granulomas with striking similarities to human-M. tuberculosis granulomas. Here, we present workflows to perform high content analyses of granulomas in adult zebrafish infected with M. marinum by cryosectioning to take advantage of strong endogenous transgenic fluorescence adapted from common zebrafish embryo infection tools. Specific guides to classifying granuloma necrosis and organisation, quantifying bacterial burden and leukocyte infiltration of granulomas, and visualizing extracellular matrix remodelling and foam cell formation are also provided. We use these methods to characterize neutrophil recruitment to M. marinum granulomas across time and find an inverse relation to granuloma necrosis suggesting granuloma necrosis is not a marker of immunopathology in the natural infection system of the adult zebrafish-M. marinum pairing. The methods can be easily translated to studying the zebrafish adaptive immune response to other chronic and granuloma-forming pathogens.

Heme oxygenase limits mycobacterial infection-induced ferroptosis

“…To determine if zebrafish reproduced this phenotype, we scored M. marinum granulomas as "organised" or "loose" based on host nuclear structure in hmox1a vcc42/vcc42 and their WT clutch mates (Fig. 2C) [2,21]. The proportion of unorganised "loose" granulomas in hmox1a vcc42/vcc42 adults was significantly higher than in the WT adults demonstrating a conserved defect in granuloma formation (Fig.…”

“…Single cell suspensions of mid log-phase fluorescent M. marinum strains and ΔESX1 M. marinum were stored at -80°C in aliquots [20]. Zebrafish infections were carried out as previously described with 200 CFU infection doses by microinjection into embryos or intraperitoneal injection into adults [20,21]. Infected adults were maintained at 28°C with 14:10 light:dark lighting cycle.…”

“…Cryosectioning was performed as previously described [21]. Hmox1 immunostaining was carried out with a mouse anti-HMOX1 primary (GeneTex GTX633693) and a goat anti-mouse Alexa Fluor 488 (Thermofisher R37120).…”

“…Imaging was carried out on Leica M205FA and DM6000B, and Deltavision Elite microscopes as previously described [17,20,21].…”

Adaptation to an amoeba host drives selection of virulence-associated traits in Vibrio cholerae

“…For the histology, dissected intestines were fixed in 10 % neutral buffered formalin overnight at 4 °C, rinsed in PBS, and then incubated in 30% (w/v) sucrose, 50:50 30 % sucrose:OCT (4583, Tissue-Tek), and OCT for ~4 h each [43]. Tissue was frozen in OCT and cryosectioned in 20 μm sections.…”