Figure 2.

mTOR deficiency impairs macrophage development and survival and sensitizes infected macrophages to mycobacterium-induced cytotoxicity

Larvae were infected with Mm expressing BFP2 (A), (E), and (I–N), mWasabi (C and D), or tdKatushka2 (F–H) fluorescent proteins via the hindbrain ventricle (A) or the caudal vein (B–N) 2 dpf.

(A) Serial confocal micrographs of granulomas in Tg(mfap4:tdTomato-CAAX) zebrafish treated with rapamycin or DMSO. Mm (cyan), macrophages (red).

(B) (Top) Macrophage counting region (shaded light blue). (Bottom) Numbers of macrophages in Mm- and mock-infected Raptor mutants and siblings expressing Tg(mpeg1:tdTomato). Symbols indicate mean values for each group. Error bars show SEM.

(C) (Top) Duration of rapamycin and DMSO treatments. (Bottom) Mycobacterial cording 5 dpi.

(D) Time-lapse confocal micrographs of a dying infected macrophage in an mtor^{sa16755/sa16755}; Tg(mfap4:tdTomato-CAAX); Tg(ubb:secA5-YFP) animal 2 dpi. Mm (blue), secreted annexin V-YFP (green), macrophage (magenta), annexin V⁺ blebs (arrowheads). See Video S1.

(E–G) 6-h time-lapse confocal microscopy of mtor^fh178/fh178 and mTOR-sufficient siblings expressing Tg(mpeg1:YFP) 2 dpi. See Video S2.

(E) Absolute numbers of infected macrophages per field.

(F) Percentage of dying infected macrophages per field.

(G) Relative mycobacterial burdens in dying macrophages of mtor^−/− and mTOR-sufficient fish. Bacterial volumes were normalized to values obtained from dying cells in mTOR-sufficient controls for each imaging run.

(H) Widefield micrograph of parabiotic zebrafish comprised of conjoined WT Tg(mpeg1:tdTomato) and mtor^fh178/fh178; Tg(mpeg1:YFP) embryos 4 dpi.

(I) Absolute numbers of macrophages in the WT body (top) and mtor^−/− body (bottom) of WT-mtor^−/− parabiont.

(J) Maximum intensity projections of infections in the WT body (top) and mtor^−/− body (bottom) of a WT-mtor^−/− parabiont 4 dpi.

(K) Widefield micrograph of WT Tg(mpeg1:tdTomato) and mtor^fh178/+; Tg(mpeg1:YFP) parabiont 4 dpi.

(L) Absolute numbers of macrophages in the WT body (top) and mtor^+/− body (bottom) of WT-mtor^+/− parabiont.

(M) Maximum intensity projections of infections in the WT body (top) and mtor^+/− body (bottom) of a WT-mtor^+/− parabiont 4 dpi.

Scale bars: 25 μm in (A), 10 μm in (D), 400 μm in (H) and (K), and 50 μm in (J) and (M). Horizontal lines indicate mean (E) and (G) or median (F) values. Statistical analyses, (E–G) two-tailed, unpaired Student’s t test. Time lapse data were pooled from five (E and F) or three (G) independent experiments.

See also Figures S1 and S2.