Fig. 4

Fig. 4

Macrophages dominate T. marneffei phagocytosis during infection establishment.

(A, B) Confocal imaging of leukocytes and calcofluor-stained acuD:RFP fungal conidia in Tg(mpx:EGFP) fluorescent leukocytes. (A) shows a germinated, RFP-expressing, blue calcofluor-stained conidium (red arrow) within a non-fluorescent vacuole of a bright EGFP^high neutrophil. Z-stack sections (i-iii) orientated as in schematic diagram. (B) shows a dull EGFP^low macrophage laden with multiple RFP-expressing conidia. (C,D) Phagocytosis of calcofluor-stained fungal conidia by a macrophage (C) and neutrophil (D) in Tg(mpeg1:mCherry/mpx:EGFP) embryo (C) inoculated in a somite. The still images are from S1 and S2 Movies respectively. The macrophage extends a long process towards the conidium (red arrowhead); conidial phagocytosis is followed by macrophage movement over the site of phagocytosis and out of the field. In each panel, maximum projection z-stack image is supplemented by xz and yz projections, orientated as in the schematic diagram. (E-G) Confocal imaging of intracellular calcofluor-stained conidia within a neutrophil (E) and macrophage (F) of a Tg(mpeg1:mCherryCAAX/mpx:EGFPCAAX) embryo, with dimensional projections as in (C). In each case, the lower right yellow-boxed panel is a detail of the yellow-boxed area in the upper left xy maximum intensity projection, displaying the cross-section (white dashed line) for which a fluorescence intensity channel profile is shown in G. Arrowheads indicate peaks of fluorescence corresponding to neutrophil (Gi, green) and macrophage (Gii, red) cellular membranes. (H) Enumeration of conidia phagocytosis (i) by macrophages (red lines) and neutrophils (green lines) reflects higher macrophage recruitment (ii) following somite infection. Lines represent individual infected zebrafish followed over time. N = 10 embryos/group imaged on n = 8 different days. P-values from Mann-Whitney test: ns = not significant, * = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001.