Rac2 is not required for macrophage migration to or phagocytosis of A. fumigatus spores. rac2+/−; mfap4::tdtomato adult fish were crossed to each other, and the resulting larvae were injected with YFP-expressing TBK1.1 (Af293) spores. Larvae were live imaged via confocal microscopy at 1 and 2 dpi and genotyped at the conclusion of the experiment. Average number of spores enumerated per larva via microscopy at 1 dpi = 60. Data represent 6 pooled replicates of 10–13 larvae per replicate. (A and B) The number of macrophages (mfap4::tdtomato) at the infection site and the number of spores present were counted, and the average number of macrophages per spore (A) and the percentage of spores found inside of macrophages (B) were calculated. Each symbol represents one larva, color coded by experiment. Lines represent emmeans ± SEM. P values calculated by ANOVA. (C) The presence of macrophage clusters was evaluated, and the percentage of larvae across all replicates with clusters is plotted. P values calculated by Fisher’s exact test. (D) Example images showing macrophage clustering around fungi. Both spore swelling (arrow) and germination (arrowhead) can be found from inside of macrophage clusters.
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