DOI: 10.5061/dryad.08803
title: Data from: Efferocytosis and extrusion of leukocytes determine the progression of early mycobacterial pathogenesis

description: Macrophages and neutrophils are the first responders to invading pathogens
 and contribute strongly to the host defense against intracellular
 pathogens. The collective interplay and dynamic interactions between these
 leukocytes are to a large extent not understood. In the present study, we
 have investigated their role using a combination of confocal
 laser-scanning and electron microscopy in a zebrafish model for
 tuberculosis, a local Mycobacterium marinum infection in the tissue of the
 larval tail fin. Our results show that neutrophils are efficient in
 phagocytosis of mycobacteria and that they contribute largely to their
 dissemination. Macrophages appear to play a major role in efferocytosis,
 phagocytosis of dead cells that contain bacterial content. Phagocytic
 cells with large bacterial aggregates are formed that can be extruded out
 of the tissue after cell death. Alternatively, these excessively infected
 cells can undergo necrosis leading to immediate recruitment of surrounding
 leukocytes and subsequent phagocytosis of released bacteria. Our data show
 that these necrotic burst events result in progression of the infection,
 whereas extrusion abates the infection.
description: Video 1. Macrophage and
 neutrophil behavior at the lag phase of Mm infection.Double transgenic
 Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were
 imaged alive every ~60 sec from 1 hpi to 12 hpi by CLSM (Nikon A1). In the
 top panel the dynamics of GFP-positive (green) macrophages and
 DsRed-positive (blue) neutrophils are shown, whereas in the bottom panels
 the trajectories for macrophages (left) and neutrophils (right) are
 indicated. The magenta and red lines represent infected macrophages and
 neutrophils, whereas the yellow and blue lines represent uninfected
 leukocytes. The infected cells remain in the tail fin, except for one of
 the neutrophils (red line), which undergoes reverse migration along the
 caudal vein. The uninfected macrophages show short trajectories in the
 tail fin and the neutrophils show longer tracks, of which one neutrophil
 (blue line) seems to be scanning a large area of the tail fin. The maximum
 intensity projection from 11 CLSM images (step size in z-direction, 5.56
 µm) is shown at 10 frames per second. Scale bar: 100
 µm.video1.mp4Video 2. Cell death Mm infected neutrophils showing fragmentation and roundup morphologies followed by efferocytosis.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). In the top panel the dynamics of DsRed-positive (blue) neutrophils are visualized separately, whereas in the bottom panels GFP-positive (green) macrophages and Mm (red) are also included. The white arrow in top panel indicates an infected neutrophil that undergoes cell death showing clear fragmentation morphology after ~1,5 hours. Subsequently, the bacteria and probably remains of this neutrophil is taken up by a second neutrophil within an hour. This second neutrophil (also indicated by an white arrow in right bottom panel) is taken up by a macrophage at 4 hours. The yellow arrow in the top panel indicates an infected neutrophil that undergoes cell death showing a round up morphology. The fluorescent signal of the neutrophil associated with Mm gradually decreases in several hours. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm.Video2.mp4Video 3. Cell death of Mm infected macrophages showing fragmentation morphology.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) indicated by white arrow undergoes cell death showing clear fragmentation at 57 minutes. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm.Video3.mp4Video 4. Cell death of Mm infected macrophages showing roundup morphology.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) undergoes cell death showing a roundup morphology at 50 minutes, and the GFP signal associated with Mm decreases gradually in 2 hours. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm.Video4.mp4Video 5. Cell death of Mm infected macrophages showing rapid signal disappearance morphologies.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) indicated by white arrow undergoes cell death showing rapid signal disappearance at 54 minutes and immediate recruitment of another macrophage. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm.video5.mp4Video 6. Macrophage burst at the exponential phase of Mm infection.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~63 sec from 1 dpi to 2 dpi by CLSM (Nikon A1). Macrophage burst and spreading of Mm (red) are shown, followed by the recruitment of GFP-positive (green) macrophages and DsRed-positive (blue) neutrophils. In the left bottom the macrophages and Mm are visualized separately. In the right bottom only the Mm are visualized, and the burst event at t = 5h is indicated by arrowheads. A neutrophil that phagocytized Mm after the burst event, indicated by an arrow in the upper panel at t= 8, is moving away from the infection site. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 50 µm.Video6.mp4Video 7. Extrusion of a Mm aggregate out of the tail fin.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~60 sec from 1 hpi to 17 hpi by CLSM (Nikon A1). The GFP-positive (green) macrophages containing a Mm (red) aggregate undergoes cell death showing fragmentation. The bacterial content is clearly extruded from the tail fin 3 hours after the macrophage showing fragmentation (Fig. 3D). The corresponding transmission channel is shown below, showing a protuberance on the outer epithelial layer, which is eventually shed off. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm.video7.mp4Video 8. CLSM on macrophage and neutrophil behavior at the exponential phase of Mm infection.Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~60 sec at 3 dpi for 2.5 hours by CLSM (Nikon A1) before fixation. The two infected GFP-positive (green) macrophages and two infected DsRed-positive (blue) neutrophils that were correlated with 3D block-face SEM images are indicated by arrows. The indicated macrophages (MP1 and MP2) phagocytized Mm approximately 2 and 1.5 hours before fixation respectively and these cells remained GFP-positive after fixation. The neutrophil (NP1) containing Mm was observed 2.5 hours before fixation and undergoes cell death, observed as rapid (within ~1 min) disappearance of the fluorescent signal, ~30 min before fixation. The second neutrophil (NP2) was recruited to another infected neutrophil ~30 min before fixation. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: right panel 100 µm and for the left panel 50 µm.video8.mp4Video 9. Three-dimensional block-face SEM images of Mm infected tail fin.A double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larva infected with M. marinum was fixed and block face SEM imaging was performed on an 80 by 80 µm region of interest using a Quanta FEG 250 with a Gatan 3View Ultramicrotome. The video shows 154 images acquired every 150 nm step size in z-direction (103 nm pixel size) at 10 frames per second. Scale Bar: 10 µm.video9.mp4Video 10. Three-dimensional block-face SEM images of necrotic neutrophil undergoing netosis.A double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larva infected with M. marinum was fixed and block face SEM imaging was performed on an 80 by 80 µm region of interest using a Quanta FEG 250 with a Gatan 3View Ultramicrotome. The video shows 70 images in z-direction (23 nm pixel size) of the neutrophil (NF2, Fig. 4). Note the partial intact nuclear envelope (arrow) and the vesicles (arrowheads) fusing with bacterial aggregate. The images were acquired with every 150 nm step size in z-direction and are in this video visualized at 10 frames per second. Scale Bar: 2 µm.video10.mp4