Orientation of the movement based on the artificial wound region.

<p>(a) Three-dimensional plot of the tracks with time as the vertical axis. The DIC image of the fish is presented as a horizontal plane at time 0 and one fluorescent slice is shown at time 120. The black square over the DIC corresponds to the artificial wound region. (b) Description of the absolute, oriented and lateral neutrophil velocities with respect to the axis defined according to a manual delineation of the wound region.</p

Hysteresis thresholding segmentation of a single neutrophil at five time points.

<p>The region described by low threshold (bright) will contain one or more regions described by the high threshold (dark); if segmented solely with a single high threshold several unconnected regions would arise. A single low threshold would in turn produce more regions of low intensity not shown here.</p

Mean neutrophil volume relative to its position in an area anterior to the wound region.

<p>The field of view was split into 25 adjacent regions each containing 20 MATLAB® columns, with higher numbers denoting neutrophils closer to the wound region. The volume was averaged in each band and normalised to the mean of the data set for each larva, which were then pooled in the final analysis. Two sets of thresholds were used. Analysis A uses higher thresholds than analysis B.</p

Validation of the algorithms with synthetic and real data sets.

<p>In all cases the sets were automatically tracked with <i>PhagoSight</i>; input thresholds were automatically determined and then modified from 40% to 140% of the original values to test the robustness against variation of that input parameter. (a) Distance from the automatically generated tracks to the gold standard (D_AG) for the synthetic data set, <i>BD</i> corresponds to the Bhattacharyya distance between background and neutrophils. (b) Distance from the gold standard to the automatically generated tracks (D_GA) for the synthetic data set. (c) D_AG for the real data set, (d) D_GA for the real data set. High distances for the synthetic sets are due to low thresholds that interpret noise as neutrophils. The increase in D_GA in (d) is caused by higher levels that do not detect faint neutrophils, this in turn will reduce D_AG as with the higher threshold, the neutrophils which are detected are the brightest and thus the tracking is more precise.</p

Segmentation of large objects in synthetic (top row) and real (bottom row) data sets.

<p>(a,d) One slice of a 3D stack where one object (box) was considered as an outlier due to its volume. (b,e) A three-dimensional rendering of the object indicates that it is formed by two neutrophils that collided. (c,f) Two new objects after the segmentation.</p

Comparison of automatic tracks against the gold standard.

<p>A synthetic data set is shown in (a) whilst (b,c) are real data sets. In (a,c) the thresholds are 140% of the automatically detected values, while for (b) they are 60% of the detected values. The automatic tracks are displayed as thick solid lines and the gold standard as thin dashed lines, and one slice of the intensity data sets is presented with the tracks. In the real data set, the high thresholds prevented the low intensity neutrophils from being detected (solid arrows) and therefore no tracks were generated for these neutrophils with corresponding high D_GA. With lower thresholds, the faint neutrophils were detected, and other neutrophils were also tracked (dashed arrow), this track could have been generated by noise or could have been missed during the manual tracking. It should be noticed that where <i>PhagoSight</i> detected the neutrophils, the tracks are very close to the gold standard.</p

Comparison of the segmentation and tracking results against ICY for one synthetic and one real data set.

<p>In ICY, “size filter” was used with increasing values of size; <i>PhagoSight</i> input thresholds were automatically determined and then modified from 40% to 140% of the original. (a) Synthetic data set tracked with ICY, (b) Synthetic data set tracked with <i>PhagoSight</i>, (c) Real data set tracked with ICY, (d) Real data set tracked with <i>PhagoSight</i>. Solid line and circle markers corresponds to distance from the automatically generated tracks to the gold standard (D_AG) and dotted line with square markers corresponds to the distance from the gold standard to the automatically generated tracks (D_GA).</p

Description of the synthetic data sets.

<p>(a) One slice at t = 26 and the paths of six neutrophils shown as coloured lines. Vertical axis indicates time. (b) Five histograms for background (noise) and neutrophils (signal) for different levels of noise. The separability is indicated by the Bhattacharyya Distance (<i>BD</i>) values: highest <i>BD</i> corresponds to more separable classes (solid lines with no markers) and lowest <i>BD</i> corresponds to less separable classes (solid lines with circle markers). (c) One slice (<i>BD</i> = 1.61, SNR = 25.2 dB) shown as a mesh, intensity corresponds to the vertical axis. (d) One slice (<i>BD</i> = 0.45, SNR = 10.7 dB). The noise can be easily compared between the two data sets.</p

Macrophages protect <i>Talaromyces marneffei</i> conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment <i>in vivo</i>

<div><p>Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen <i>Talaromyces marneffei</i> and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established <i>Aspergillus fumigatus</i> model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions <i>in vivo</i>. Following parenteral inoculation, <i>T</i>. <i>marneffei</i> conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in <i>T</i>. <i>marneffei</i> conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new <i>in vivo</i> model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting <i>T</i>. <i>marneffei’s</i> opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.</p></div

Neutrophils and macrophages play opposing roles during establishment of <i>T</i>. <i>marneffei</i> infection.

<p>(A) Representative images of 52 hpf <i>Tg(mpeg1</i>:<i>mCherry/mpx</i>:<i>EGFP)</i> embryos injected with antisense morpholino oligonucleotides to perturb the balance of neutrophil and macrophage populations. (B) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi corresponding to the aligned treatment groups in panels (Ai-iv), for wild-type (WT) and myeloperoxidase-deficient (<i>mpx</i>^-/-) genotypes as shown. Data are mean±SEM for n≥3 experiments, n≥5 embryos/group/experiment.</p