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

<i>T</i>. <i>marneffei</i> infection of zebrafish at 28°C.

<p>(A-C) Histological time-course following <i>T</i>. <i>marneffei</i> inoculation of zebrafish, stained by Grocott methanamine silver and Evan’s Blue counterstain (i), with adjacent hematoxylin and eosin-stained sections (ii). mpi, minutes post infection; dpi, days post infection. Red arrowheads indicate fungal conidia. White arrowheads indicate Duct of Cuvier (A) and leukocyte with intracellular fungal elements (C). (D) <i>T</i>. <i>marneffei</i> CFU time-course at 28°C following intravascular inoculation of target dose of approximately 150 fungal conidia (actual dose verified by 0 dpi CFU). Different colors indicate 3 independent experiments, mean±SEM, n≥5 embryos/group/experiment. *p = 0.016, **p = 0.0059, ***p = 0.0003 for statistical comparison between 3 and 4 dpi. (E) Embryo survival following intravascular inoculation in (D). Data are pooled embryos from 3 experiments: n = 474 uninfected, 339 infected. NS: not significant.</p

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

<i>T</i>. <i>marneffei</i> infection of zebrafish at 33°C.

<p>(Ai-iv) Histology of infected zebrafish embryos at different stages of <i>T</i>. <i>marneffei</i> infection. Sections were stained with Grocott methanamine silver to visualize fungi and counterstained with Nuclear Fast Red. Scale bar: 10 μm. (i) Phagocytosed conidium (arrowhead) within host leukocyte in the circulation at 20 minutes post infection. (ii) At 2 dpi, two fungal morphologies were observed: intracellular fungi assumed typical yeast morphology (upper arrowhead), while extracellular fungi displayed an elongated form (lower arrowhead). (iii) Yeast morphology of <i>T</i>. <i>marneffei</i> within a circulating infected leukocyte (arrowhead) at 3 dpi. (iv) Granuloma formation at 4 dpi. (B)FACS-purified, cytospun, <i>T</i>. <i>marneffei</i>-laden leukocytes prepared from infected embryos at 4 dpi, incubated at 33°C during infection. Grocott methanamine silver with Nuclear Fast Red counterstain. (i and ii) show neutrophils laden with hyphal forms (empty arrowheads), representative of all neutrophils observed. (iii) shows two macrophages laden with yeast forms (full arrowheads), the dominant morphology observed. (iv) shows a macrophage containing fungal cells of both yeast and hyphal morphologies. Scale bar: 10 μm. (C) Three representative CFU time-courses of infection at 33°C. In contrast to infections at 28°C (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.g001" target="_blank">Fig 1D</a>), no consistent decrease in CFU was observed at 4 dpi. Data are mean±SD of 3 independent replicates, n = 5 embryos (pooled)/timepoint/experiment. (D) Kaplan-Meier life table analysis for the treatment groups shown in (E). Significantly reduced survival occurred in infected groups compared to uninfected groups. n = 297 for uninfected, 319 for infected. P-value from Gerhan-Breslow-Wilcoxon Test. (E) Measurement of neutrophil populations during infection using Neutrophil Units. A significant increase in neutrophil population size occurred in the infected group compared to uninfected group over 1–4 dpi. Data are mean+SEM from 3 independent experiments. n = 5 embryos/group/timepoint in each experiment. P-values from unpaired two-tailed t-test.</p

Macrophages dominate <i>T</i>. <i>marneffei</i> phagocytosis during infection establishment.

<p>(A, B) Confocal imaging of leukocytes and calcofluor-stained acuD:RFP fungal conidia in <i>Tg(mpx</i>:<i>EGFP)</i> 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 <i>Tg(mpeg1</i>:<i>mCherry/mpx</i>:<i>EGFP)</i> embryo (C) inoculated in a somite. The still images are from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s012" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s013" target="_blank">S2</a> 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 <i>Tg(mpeg1</i>:<i>mCherryCAAX/mpx</i>:<i>EGFPCAAX)</i> 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.</p

Zebrafish mount a Csf3r-dependent granulopoietic response to <i>T</i>. <i>marneffei</i> infection.

<p>(A) Low magnification images of infected and uninfected whole <i>Tg(mpx</i>:<i>EGFP)</i> embryos at 0, 3 and 4 dpi, displaying the profound infection-driven increase in EGFP-expressing neutrophil numbers. (B, C) Neutrophil (B) and macrophage (C) population expansion in infected versus uninfected embryos from 0–4 dpi. These data derive from enumerating neutrophil and macrophage numbers in the same embryos, of genotype <i>Tg(mpx</i>:<i>EGFP/mpeg1</i>:<i>Gal4FF/UAS-E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i>. Data displayed are mean±SD, n = 4 embryos/group. These data replicate other data derived from separately scoring these leukocyte populations in different embryos, in n≥2 independent other experiments. (D) Neutrophil population sizes at 4 dpi represented by Neutrophil Units in embryos injected with antisense morpholino oligonucleotides targeted to knockdown <i>csf3</i>-receptor (<i>csf3r</i>-MO) or interleukin-6 receptor (<i>il6ra</i>-MO and <i>gp130</i>-MO) Data are mean±SEM, n = 10 embryos/group/experiment, n≥3 experiments. Intragroup +/- infection p-values from 2-tailed unpaired t-test; p-values from intergroup comparisons involving <i>csf3r</i>-MO from 1-tailed unpaired t-test, given the <i>a priori</i> expectation of a reduction in neutrophil values in <i>csf3r</i>-deficiency [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.ref065" target="_blank">65</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.ref095" target="_blank">95</a>] (E) Kaplan-Meier life table analysis for the treatment groups shown in (C). Numbers/group (uninfected/infected): Control MO, 474/326; <i>csf3r</i>-MO, 443/233; <i>il6ra</i>-MO, 462/330; <i>gp130</i>-MO, 465/313. P-values from Gerhan-Breslow-Wilcoxon Test.</p

Depletion of the macrophage niche increases conidial destruction during establishment of infection.

<p>(A) Ablation of macrophages decreases conidial viability in the first 24 hpi. (i) and (ii) show representative images of nitroreductase-dependent metronidazole-mediated macrophage ablation following treatment of transgenic embryos (ii) versus untreated control embryos (i). Ablation efficiency is quantified in (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s010" target="_blank">S9A Fig</a>). (B) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi in macrophage-replete control (<i>Tg(mpeg1</i>:<i>Gal4FF/UAS-E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> negative, treated with 10 mM metronidazole) compared to macrophage-depleted (<i>Tg(mpeg1</i>:<i>Gal4FF/ UAS- E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> positive, treated with 10 mM metronidazole). (C) Ablation of neutrophils increases conidial viability in the first 24 hpi. (i) and (ii) show representative images of Nitroreductase-dependent metronidazole-mediated neutrophil ablation in treated (ii) versus diluent-treated control embryos (i). Ablation efficiency is quantified in (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s010" target="_blank">S9B and S9C Fig</a>). (D) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi in neutrophil-replete control (<i>Tg(mpx</i>:<i>KalTA4/UAS-E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> negative, treated with 10 mM metronidazole) compared to neutrophil-depleted (<i>Tg(mpx</i>:<i>KalTA4/ UAS- E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> positive, treated with 10 mM metronidazole) Data are mean±SEM, n≥5 embryos/group/experiment, n≥3 experiments.</p