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. 2018 May 30;20(9):e12858. doi: 10.1111/cmi.12858

Figure 2.

Figure 2

Wild type M. marinum can still infect brain tissue when macrophages are depleted. Left panel shows untreated zebrafish larvae (control), whereas right panel shows larvae depleted of phagocytes by treatment with pu.1 and clodronate filled liposomes at 3 dpf, to kill circulating phagocytes. (a) Control casper larva at 5 dpf, stained with anti‐L‐plastin to visualise normal phagocyte distribution. (b) Five dpf phagocyte‐depleted casper larva. Anti‐L‐plastin is used in (a) and (b) to stain phagocytes. (c) Dorsal view of wild type Tg(Fli1:GFP) y1 larvae (green) at 5 dpi, systemically infected at 4 dpf with M. marinum (red) and stained with anti‐L‐plastin (cyan), showing formation of early granuloma in brain tissue. (d) Z‐stack of boxed area in (c), allowing for a more precise analysis of the position of M. marinum and phagocytes. (e) Corresponding red fluorescent channel to clearly show infection pattern. (f) In the presence of macrophages, M. marinum leaves the bloodstream within phagocytes (arrow) and forms early granulomas in brain tissue. Scale bar c = 100 μm. Scale bar d–f = 25 μm. (g) Z‐stack of head of Tg(Fli1:GFP) y1 larva, phagocyte‐depleted, and systemically infected with M. marinum (red) at 4 dpf and stained with L‐plastin (cyan). Boxed area is enlarged in h–j. (h) Shows mycobacteria outside blood vessels in brain tissue in the absence of phagocytic cells (*). (i) Shows the corresponding red fluorescent channel, depicting that tissue infiltration follows the shape of the blood vessels. (j) Single Z‐slice, which shows the intracellular phenotype of M. marinum (arrow), colocalising with a blood vessel, but in the absence of L‐plastin labelling. Scale bar g = 100 μm; Scale bar h–j = 25 μm. (k) Schematic representation of pooled data of all early granulomas found in nine wild type larvae showing a random distribution in the brain. (l) Schematic representation of pooled data of infection distribution found in nine phagocyte‐depleted larvae, showing that mycobacteria are found in brain tissue, but do not migrate into deeper tissue