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

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 Talaromyces marneffei 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 Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei 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 T. marneffei 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 in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei’s opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.


Introduction
macrophages also may play a role in pathogen dissemination [14]. Many fungal pathogens, such as H. capsulatum and T. marneffei, proliferate within macrophages as yeast [15][16][17], while invasive hyphae formed by A. fumigatus cannot be phagocytosed and elicit a neutrophil-dominated response, including generation of reactive oxygen species and formation of neutrophil extracellular traps (NETs) [18]. Although much has been learnt from mammalian infection models regarding disease progression, these models are inherently limited with regards to observing host-pathogen interactions in vivo and in real-time.
Here we present a new in vivo zebrafish model of talaromycosis (formerly called penicilliosis) that is caused by T. marneffei, a thermally dimorphic, opportunistic pathogen of humans. T. marneffei is capable of switching between a saprophytic hyphal growth form and a pathogenic yeast form in response to temperature and host cues including factors such as pH, salt concentration, calcium signalling and iron availability [40][41][42][43]. In the host, it primarily occupies the phagocyte niche but extracellular fungal cells are also evident, presumably due to host cell death.
For these studies, we have exploited the ectothermic nature of the zebrafish host. Although zebrafish are customarily held at 28˚C in the laboratory, their normal development is documented for temperatures up to 33˚C [44], and in the wild populations are found at temperatures up to 38.6˚C [45]. We model an invasive hyphal form of T. marneffei infection unique to this zebrafish model (at 28˚C) and a disseminated intracellular yeast form of infection resembling the human disease (at 33˚C). These studies show that thermal dimorphism can be overridden by intracellular cues. We demonstrate a protective, G-CSFR-dependent expansion of neutrophils during protracted infection. Our studies particularly focused on the initial period of infection establishment, when fungal spores first encounter host leukocytes. These studies show that macrophages provide a protective niche for fungal conidia during infection establishment, whereas neutrophils exhibit a strongly fungicidal activity towards conidia that is myeloperoxidase dependent.

Intravascular delivery of T. marneffei establishes a reproducible systemic invasive filamentous form of talaromycosis in zebrafish larvae at 28˚C
To optimise modelling of talaromycosis in zebrafish larvae, a variety of pathogen inoculation approaches and larval culture conditions were tested. At 28˚C, immersion of 24 hpf embryos in T. marneffei conidia delivered within the chorion did not result in infection (0% (0/55) infected and 2% (1/56) death after 24 hours of co-incubation). However, reproducible local infection was established by intramuscular or 4 th ventricle injections, while systemic infection was initiated by intravenous inoculation into the Duct of Cuvier at 52 hpf. Histology demonstrated fungal conidia in close proximity to vascular walls immediately following inoculation (Fig 1A), their immediate phagocytosis by leukocytes ( Fig 1B) and their germination within 1 day post infection (dpi), including within intravascular leukocytes (Fig 1C). From these initial dose-finding studies, it was established that intravenous inoculations of 100-150 viable conidia established a systemic infection that resulted in <25% mortality during the course of the experiment, with stable fungal colony-forming unit (CFU) counts until 4 dpi, when CFU counts declined (Fig 1D and 1E). This indicates that for infective challenges in this dose range, the zebrafish innate immune system alone is capable of controlling a T. marneffei infection. As expected, considering the thermal dimorphism of T. marneffei [3], during the later days of infection extension of fungal germ tubes within phagosomes elongated some leukocytes ( Fig  1C and S1D Fig). In some embryos, there was also invasive filamentous growth within various tissues (S1C and S1H Fig), including the brain (S1E Fig). By 3-4 dpi, accumulation of leukocytes at infection foci resulted in abscess and/or a tight collection of cells reminiscent of the "early granuloma" observed in larval M. marinum infection [46] (S1C and S1F-S1H Fig).
Although this form of invasive filamentous infection digressed from the predominantly yeastform infection observed in human talaromycosis, it did confirm that in vivo infection per se did not provide sufficient cues for T. marneffei to switch to its yeast form.

The intracellular environment of leukocytes can override thermal T. marneffei dimorphism signals at 33˚C
To model infection with yeast morphology T. marneffei, as observed in human disease [47], infection was also established at 33˚C [3] (Fig 2). This is a temperature which zebrafish tolerate (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 T. marneffei within a circulating infected leukocyte (arrowhead) at 3 dpi. (iv) Granuloma formation at 4 dpi. (B)FACS-purified, cytospun, T. marneffeiladen 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 (Fig 1D), 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. well [26,29,44,[48][49][50][51], and at which the yeast morphological switch occurs in vitro [40]. Furthermore, it reflects the temperatures of human torso and extremity skin (31-34˚C) [52][53][54], a tissue characteristically involved in disseminated talaromycosis and where intracellular yeast forms are readily found on biopsy [55,56].
Despite an extensive literature of zebrafish experimentation at 33˚C, we specifically verified experimentally that zebrafish phagocyte function is intact at 33˚C. We conducted comparative experiments at 28˚C and 33˚C with Aspergillus fumigatus, a fungal pathogen that is not thermally dimorphic, and has been previously studied in zebrafish at 28˚C [32-35]. We compared four parameters of phagocyte function at the two temperatures: (1) the initial migratory response of neutrophils and macrophages to the site of conidial inoculation (S2A Fig); (2) the initial phagocytic response of neutrophils and macrophages to A. fumigatus conidia following their arrival at the site of conidial inoculation (S2B Fig); (3) the myelopoietic response to inoculation with A. fumigatus conidia over a 4-day period (S3 Fig); (4) the impact of morpholino-induced perturbations of leukocyte abundance on the germination of A. fumigatus 24 hpi (S4 Fig). Furthermore, for scenarios (1-3), we conducted these experiments with both live conidia and dead conidia (killed by γ-irradiation) to address the possibility that, had any variation been observed between temperatures, that this might be due to different rates of conidial germination and/or proliferation at the two temperatures. For each of these 13 experimental scenarios, no consistent significant difference was observed in any endpoint (S2-S4 Figs). 7/53 pairwise comparisons testing the null hypothesis "that there was no temperature-dependent difference in the endpoint" generated a p-value (corrected for multiple comparisons) that was <0.05. We therefore concluded that phagocyte numbers and function were intact at 33˚C and that experiments conducted at 33˚C would make a valid contribution to an exploration of the role of phagocyte function in the pathogenesis of T. marneffei infection establishment.
For infections that proceeded at 33˚C, histology again demonstrated leukocyte conidia phagocytosis (Fig 2Ai) and T. marneffei growth within leukocytes and tissues (Fig 2Aii-2Aiv). Cytospun leukocyte preparations at 4 dpi of 33˚C infections demonstrated germinated, proliferating fungal cells within leukocytes. Strikingly, at this temperature, T. marneffei assumed an elongated, septate filamentous form within all observed neutrophils, whereas within macrophages, fission yeast morphology (recognized as characteristic elongated forms with a medial septum [57]) predominated (7/9 macrophages) ( Fig 2B). We confirmed that also in mammalian macrophages at 33˚C, T. marneffei assumed this characteristic fission yeast morphology (S5 Fig). Collectively, these morphologically-based observations indicate that the different intracellular milieu of each phagocyte type can differentially influence T. marneffei form, and that at 33˚C, the macrophage milieu favours the transition to the yeast form.
At 33˚C, despite a vigorous granulopoietic response to infection (Fig 2E), fungal CFU numbers did not decrease at 4 dpi (Fig 2C), and death from infection was increased (Fig 2D), suggesting enhanced fungal viability at this temperature.

Sustained talaromycosis triggers a protective myelopoietic response
To facilitate live imaging of host-pathogen interactions, infection was performed using larvae expressing neutrophil-and/or macrophage-specific fluorescent transgenes. Quantification of leukocyte numbers over 4 days of infection at 28˚C revealed a dramatic increase from 2-4 dpi in both neutrophil and macrophage numbers in response to active infection (Fig 3A-3C). By 4 dpi, macrophage and neutrophil numbers had increased to levels 150% and 260% respectively of those in uninfected controls, while pathogen numbers decreased by 65% over the same period (Fig 1D), indicating that the vigorous myelopoietic response was a component of an effective host response that controlled the infective burden. A vigorous granulopoietic response was also seen over 4 days of infection at 33˚C (Fig 2E).

The protective granulopoietic response is CSF3R (G-CSFR)-dependent
Amplification of leukocyte numbers during zebrafish bacterial infection has been shown to depend on signalling through the Csf3-Csf3r pathway [58], while both Csf3 and Interleukin-6 are known to be important in the response to yeast infection in mammalian systems [59,60]. To interrogate these pathways in the context of talaromycosis, Csf3 signaling was intercepted by knocking down the single chain of the homodimeric csf3 receptor (csf3r), and interleukin-6 signalling was intercepted by knockdown of each subunit of the heterodimeric interleukin 6 receptor (il6ra and gp130). To quantify granulopoiesis, infections were performed in Tg(mpx: EGFP) larvae at 28˚C and quantified as previously described [61].
Csf3r knockdown significantly reduced baseline neutrophil numbers and also significantly reduced the increase in neutrophil population size at 4 dpi ( Fig 3D). The relative increase in neutrophil abundance in both control and csf3r-knockdown infected embryos was 2.2-fold. Hence the significant difference in the absolute increase is likely to have in part reflected the significantly lower basal neutrophil abundance in csf3r-knockdown embryos. This difference was not due to a different pathogen burden following infection establishment in the face of lower basal neutrophil abundance, since csf3r knockdown did not alter fungal survival/proliferation as reflected by 24 hours post infection (hpi) CFU numbers ( Fig 5B). By 4 dpi, the significantly reduced granulopoietic response of infected csfr3-knockdown embryos resulted in impaired survival from infection ( Fig 3E).
In contrast, il6r subunit knockdown did not impair the infection-driven granulopoietic response ( Fig 3D). The effect of il6r subunit knockdown on basal neutrophil population size was also modest.
Collectively, these data indicate that the large expansion of neutrophil numbers during sustained T. marneffei infection is dependent on an interaction between pathogen and host, which mounts a cytokine-driven granulopoietic response that is in part csf3r-but not il6rdependent. Intact basal csfr3 signalling is required for effective, protective, host-defences to talaromycosis.

Phagocytosis of conidia during infection establishment is dominated by macrophages
Since leukocyte/conidia interactions were a prominent histological feature of the initial host response to T. marneffei conidia inoculation, they were examined in detail at 28˚C (Fig 4). For these experiments, we used intramuscular inoculations, as we were not testing hypotheses about the normal route of infection, but rather about the direct interaction between conidia and leukocytes. These studies were conducted at 28˚C, as the conidial state of T. marneffei at the time of inoculation is not temperature dependent, and this is the temperature at which zebrafish leukocyte function is usually characterised. ±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 csf3-receptor (csf3r-MO) or interleukin-6 receptor (il6ra-MO and gp130-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 csf3r-MO from 1-tailed unpaired t-test, given the a priori expectation of a reduction in neutrophil values in csf3r-deficiency [65,95]   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 Tg(mpeg1:mCherry/mpx:EGFP) embryo (C) inoculated in a somite. The still images are from S1 and S2 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 Tg(mpeg1:mCherryCAAX/mpx:EGFPCAAX) 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. We confirmed that leukocytes actually phagocytosed fungal conidia, rather than merely associated with them. Fig 4A shows a neutrophil with a calcofluor-stained, germinated conidium, proven by z-stack analysis to be within an intracellular vacuole. Fig 4B shows a GFP-low macrophage in the Tg(mpx:EGFP) line (as previously described [62]) containing multiple germinated conidia. Using reporter lines in which red and green fluorophore expression is driven in macrophages and neutrophils respectively, active interaction of both neutrophils and macrophages with calcofluor-stained conidia was documented in the initial stages of infection ( Fig  4C and 4D and S1-S3 Movies), with apparent phagocytosis. Using new reporter lines in which reporter fluorophore expression was targeted to membranes by linkage to the CAAX prenylation signal, Z-stack profiling confirmed that conidia associated with leukocytes were intracellular and within membrane-lined phagosomes, as demonstrated by cross-sectional fluorescence intensity profiles (Fig 4E-4G).
Although both neutrophils and macrophages were capable of phagocytosing T. marneffei conidia, following both somitic ( Fig 4G) and intravascular inoculation (S6 Fig), T. marneffei conidia were phagocytosed almost exclusively by macrophages (Fig 4H). To quantify the various leukocyte-pathogen interactions at the inoculation site, fluorescence signal colocalization were analyzed based on the different leukocyte-associated reporter fluorophores and blue fluorescence of calcofluor-labelled conidia. Analysis of the caudal hematopoietic tissue of Tg(mpx: EGFP/mpeg1:mCherry) embryos 2 hours following intravenous calcofluor-labelled conidia inoculation showed that, while the neutrophil:macrophage voxel ratio was 1:2.6 (S6A Fig), reflecting the relative population sizes of the two phagocyte types, the ratio of conidia associated with these neutrophils and macrophages was 1:60 (S6B Fig). In contrast, following intramuscular inoculation, although T. marneffei conidia were still preferentially phagocytosed by macrophages, neutrophils also actively engaged in conidial phagocytosis (Fig 4Hi). Hence, although both neutrophils and macrophages phagocytose T. marneffei conidia during the initial stages of infection, macrophage phagocytosis predominates.

During infection establishment, neutrophils are fungicidal, while macrophages provide a protective niche
Despite a common function as professional phagocytes, neutrophils and macrophages utilize distinctive antimicrobial arsenals [63], and hence display diverse antimicrobial properties that are often microbe-specific. Given the specific effects of the neutrophil and macrophage intracellular milieu on T. marneffei form at later stages of infection (Fig 2B), we hypothesized that neutrophils and macrophages might also play divergent roles during infection establishment.
To determine the different roles of neutrophils and macrophages during the initial stages of T. marneffei infection, CFU counts were compared at 0 and 24 hpi in embryos at 28˚C that had been experimentally manipulated to modulate the relative numbers of neutrophils and macrophages (Fig 5).
Viable fungal cell number did not change between 0 and 24 hpi in leukocyte-replete control embryos (109.7% of baseline on average at 24 hpi) (Fig 5Ai and 5B), indicating either that conidia are both non-proliferative and resistant to killing, or that fungal death and proliferation were balanced in this context.
In embryos depleted of both neutrophils and macrophages by spi1+csf3r-knockdown using antisense morpholino oligonucleotides [64], 24 hpi T. marneffei CFU counts were significantly reduced to 64.4±6.7% of baseline (Fig 5Aiii and 5B). This demonstrated both that the presence of leukocytes during infection establishment enhanced conidial survival and/or fungal proliferation, and indicated the existence of a leukocyte-independent fungicidal activity.
In csf3r morphant embryos, which are depleted of neutrophils with little effect on macrophages [65,66], 24 hpi CFU counts were 101.8±10.3% of baseline (Fig 5Aii and 5B), indicating that the presence of normal numbers of macrophages alone is sufficient to protect T. marneffei conidia from the leukocyte-independent fungicidal activity observed in spi1+csf3r morphants.
To complement the phagocyte depletion experiments, the effects of expansion of neutrophil populations on T. marneffei viability was examined in scenarios of different macrophage abundance. As previously reported [67], overexpression of csf3b from mRNA injection relatively selectively increased neutrophil numbers. In our hands, it resulted in approximately twice as many neutrophils at 2 dpf (S7Ai and S7Aii Fig) compared to controls, but only an increase of 39% in macrophages (S7Aiii and S7Aiv Fig) [68]. No significant change in fungal viability was observed over the first 24 hpi for embryos overexpressing csf3b (S7B Fig), indicating that the slightly increased macrophage population present in these embryos is sufficient to provide for conidial viability, even when neutrophil populations are expanded.
Knockdown of irf8 expands neutrophil numbers, but at the expense of macrophage numbers [61,69]. In irf8 morphants, CFU counts at 24 hpi were 37.1±6.7% of baseline, which was significantly lower than for both control animals and spi1+csf3r morphants (Fig 5B), pointing to a potent fungicidal activity of the expanded neutrophil population when availability of the protective macrophage niche is reduced.
Collectively, all four scenarios support the hypothesis that macrophages provide a protective niche for T. marneffei conidia during infection establishment, shielding conidia from both neutrophil-dependent and leukocyte-independent fungicidal mechanisms.
In agreement with the important role played by neutrophils in controlling infection at later timepoints (Fig 3E), neutrophils were found to be strikingly fungicidal. As neutrophils were clearly the more fungicidal leukocyte during establishment of T. marneffei infection, we examined potential mechanisms that might determine neutrophil response to this pathogen and its outcome.

Neutrophil fungicidal activity requires myeloperoxidase
Myeloperoxidase is an abundant neutrophil enzyme important for generating potent antimicrobial radicals, and is critical for defence against other fungal pathogens in mammals [70][71][72]. We therefore hypothesized that neutrophil-dependent fungicidal activity against T. marneffei was dependent on myeloperoxidase activity. We tested the requirement for myeloperoxidase (mpx) using an mpx-deficient zebrafish mutant that is neutrophil-replete but lacks enzymatic Mpx activity [73].
During the establishment phase of infection, there was no difference in T. marneffei CFU counts at 24 hpi between WT and mpx -/larvae for infections at both 28˚C and 33˚C (Fig 5B  and S8A Fig) despite equivalent neutrophil populations over this period (S8B Fig). However, the enhanced fungicidal activity of the expanded neutrophil population in irf8-MO embryos, which reduced CFU counts at 24 hpi, was lost in mpx -/embryos (Fig 5B).
During sustained infection, mpx -/embryos mounted a vigorous granulopoietic response which is >2.5-fold higher than that of WT embryos at 3 dpi (S8B Fig). However, despite this expanded neutrophil population, mpx -/embryos carried a fungal CFU burden similar to WT embryos (S8A Fig).
Collectively, these data indicate that the fungicidal activity of neutrophils against T. marneffei is in part myeloperoxidase-dependent, and that this component of neutrophil antifungal activity becomes most critical when the neutrophil population is expanded and macrophage abundance is depleted.

Depleting the macrophage niche enhances clearance of conidia during infection establishment
During infection establishment, our results support a model in which T. marneffei preferentially parasitizes macrophages, which provide an intracellular niche that shields conidia from both neutrophil-dependent and neutrophil-independent anti-fungal mechanisms. We therefore hypothesized that limiting access to the macrophage niche would enhance conidial clearance during infection establishment.
Conversely, selective ablation of neutrophils by metronidazole treatment of Tg(mpx: KalTA4/UAS-E1b:Eco.nfsB-mCherry) embryos resulted in a 20% increase in fungal viability compared to controls (Fig 6C and 6D, S9B and S9C Fig), further supporting our findings that neutrophils exhibit fungicidal properties during infection establishment.
Taken together, these results suggest that limiting access to the macrophage intracellular niche may be one targetable pathway for restricting establishment of infection for the benefit of the host.

Discussion
This new model of Talaromyces marneffei infection in larval zebrafish holds many advantages over current murine in vivo models [76,77]. It combines a replete vertebrate innate immune system with transgenically labelled lineages and optical transparency to facilitate detailed live imaging of host-pathogen interactions. Additionally, the ectothermic biology of zebrafish allows experimental separation of thermal and host-dependent influences on fungal morphology. While this can also be achieved using the recently developed Galleria mellonella and Caenorhabditis elegans models [78,79], the invertebrate hemocyte provides only limited insight into complex innate immune responses such as those described here.
Combining an ectothermal host and a thermally dimorphic pathogen in an experimental system modelling a human infection provides both opportunity and raises some technical questions. Although zebrafish experiments are normally conducted at 28-28.5˚C, zebrafish physiology is robust at 33˚C. Evidence for this is: existence of wild D. rerio populations in waters up to 38.6˚C [45]; normal embryological development over this temperature range [44]; experimental studies involving human xenotransplantation [49]; conditional temperature sensitive mutant alleles [26, 50,51]; and even temperature shifts employed in previous infection modelling studies [26,29]. We provide new experimental evidence showing that the myeloproliferative and functional phagocyte response to A. fumigatus, a non-thermally dimorphic fungal pathogen, is similar at 28˚C and 33˚C.
T. marneffei exhibits thermal dimorphism, but even so, the fungal to yeast shift is not absolute at 37˚C. At 37˚C in vitro, the rate of hyphal conversion to yeast varies from 40% to 90% between the lowest and highest tolerated pHs, and NaCl concentrations !4% suppress hyphal to yeast conversion almost completely [40]. Even in human infection at 37˚C, extracellular forms characteristically assume an elongated shape [47,80]. As is uniquely possible in an ectothermic model, we exploited the ectothermic biology of the zebrafish host to identify a divergence between fungal morphology and temperature, specifically in response to the leukocyte intracellular milieu. The phase transition to a yeast morphology during human T. marneffei infection at 37˚C is widely regarded to be triggered primarily by the ambient temperature, with the thermal dimorphism of the pathogen seen as an evolved pathogenic trait [42]. At 33˚C, we observed filamentous forms in neutrophils and tissues (Fig 2Aii, 2Aiv, 2Bi and 2Bii), and predominantly yeast forms within macrophages (Fig 2Biii and 2Biv), providing strong evidence that the intracellular milieu of the phagocytosing leukocyte type may be a determinant of T. marneffei form in vivo that can override the influence of ambient temperature. We therefore hypothesize that one factor contributing to the macrophage being the preferred infectious niche for T. marneffei is its temperature-independent support of a phase transition to the more pathogenic yeast form.
In response to prolonged infection, we observed expansion of leukocyte populations, particularly neutrophils, and demonstrated that this protective response was at least in part dependent on signalling through G-CSFR. This demand-driven hematopoietic response to infection appears to be conserved, and has been since reported by others in response to Salmonella enterica infection [58]. Overexpression of the zebrafish Csf3r receptor ligand Csf3b by mRNA overexpression phenocopied the granulopoietic response observed during T. marneffei infection, further supporting our findings.
Genetic manipulation of leukocyte lineage numbers prior to infection demonstrated that macrophages provide a protective niche for fungal conidia, while neutrophils are fungicidal. Further, using an existing mutant, we demonstrated that the fungicidal activity of neutrophils was myeloperoxidase-dependent. Myeloperoxidase deficiency is a prevalent congenital human disorder of neutrophils with an incidence of 1:2000 [81]. Epidemiological studies associate myeloperoxidase-deficiency with increased risk of fungal infection [82], myeloperoxidase-deficient mice are vulnerable to Candida infection [83], and myeloperoxidase-deficient neutrophils display reduced ability to produce fungicidal neutrophil extracellular traps (NETs) [84]. Our demonstration of myeloperoxidase deficiency as a talaromycosis disease-enhancer in zebrafish suggests it may be an unrecognized disease-modifier of human talaromycosis and invites an evaluation of myeloperoxidase status in such patients.
Selective depletion of macrophages using the metronidazole system demonstrated that removal of this protective niche exposes T. marneffei conidia to neutrophil-dependent and independent antifungal mechanisms. Temporary depletion of macrophages in patients that transiently selectively reduce access of conidia to the macrophage niche might provide a novel therapeutic strategy to restrict infection establishment. We also hypothesize that transient macrophage ablation therapy in established talaromycosis may facilitate talaromyces infection eradication by exposing the organism to fungicidal neutrophils.
One limitation of this zebrafish model is that the infection is established by inoculation, rather than via alveolar macrophages which is presumed to be that natural route of infection [85]. However, in human T. marneffei infection, fungal forms are found in tissue macrophages throughout the body, including particularly in skin [56], lymph nodes [86] and bone marrow [87], and so our observations of the behaviour of tissue macrophages provide relevant insights to understanding the pathogenesis of the human disease.
Findings from this new in vivo zebrafish T. marneffei infection model have implications not only for talaromycosis but also for the pathogenesis of infections with other fungal and dimorphic pathogens including histoplasmosis, blastomycosis and coccidioidomycosis. Furthermore, it is a unique resource for exploring in detail the in vivo cell biology of leukocytepathogen interactions during this infection.

Ethics and biosafety statement
Animal experiments followed appropriate NHMRC guidelines and were conducted under protocols approved by Ethics Committees of the Walter and Eliza Hall Institute (2007.004 and 2009.031) and Monash University (MAS/2010/18). In accordance with the approved protocol requirements, all zebrafish embryos and larvae used in experiments were younger than 7 dpf (i.e. experiments were concluded on the 6 th dpf which was the 4 th day after infection). We performed the experiments under Institution Biosafety Committee Notifiable Low Risk Dealing (NLRD) approvals 2007.01 (Walter and Eliza Hall Institute) and PC2-N23-10 (Monash University). T. marneffei was assigned to Risk Group 2 at the time these approvals were granted. In most jurisdictions, including endemic regions, T. marneffei is a risk group 2 organism.

Talaromyces marneffei
T. marneffei strains, derived from the FRR2161 type strain, were: acuD:RFP strain, which expresses RFP on germination and the control strain SPM4 [90]. To prepare cells for injection, T. marneffei conidia were inoculated onto Sabouraud Dextrose (SD) medium and cultured at 25˚C for 10-12 days when the cultures were conidiating. Conidia were washed from the plate with 0.005% Tween 80 solution, filtered, sedimented (6000 rpm, 10 min), resuspended in dH 2 O and stored at 4˚C. For inoculation, conidia were resedimented and resuspended in PBS. Heat-inactivation and calcofluor staining was as described previously [22]. Calcofluor staining did not affect conidial viability, as evidenced by their subsequent germination in vivo (S1A Fig). T. marneffei colony forming unit (CFUs) numbers per embryo were determined by thorough homogenization of individual embryos in 500 μL of dH 2 O using a Dounce homogenizer. 250 μL of homogenate was cultured on SD medium agar with 1% ampicillin for 3-4 days at 37˚C. Plates were then incubated overnight at room temperature, and colonies that underwent yeast to hyphal morphological switching were scored as T. marneffei colonies.

Zebrafish infection with Talaromyces marneffei
For inoculation, 52 hpf tricaine-anesthetized embryos were mounted on an agar mould with head/yolk within the well and tail laid flat on the agar. The T. marneffei conidial suspension was inoculated using a standard microinjection apparatus and large-bore needle via the common cardinal vein for systemic infection, or the 4 th ventricle or a somite aligned to the yolk extension tip for local infection [22,91]. Inoculated embryos were held at 28˚C or 33˚C according experimental design. The delivered conidial dosage was determined by immediate CFU enumeration on a group of injected embryos. Following initial dose-finding experiments that established an intravascular inoculum of 100-150 CFU/embryo achieved <25% mortality for 28˚C infections (Fig 1D and 1E), this was the target inoculum dose.

Zebrafish infection with Aspergillus fumigatus
Freshly-prepared A. fumigatus conidia stocks (strains CEA10 and 295) for these experiments were stored at 4˚C for < 2 months. Fresh aliquots were prepared for microinjection as described for T. marneffei spores, delivering a target inoculum of 50-150 live spores, verified by back-plating as described above for T. marneffei. To prepare dead A. fumigatus conidia for microinjection, they were γ-irradiated with 10kGy (delivered over 207 hr 27 min 10 sec) from the Monash University Gammacell 40 Exactor (Theratronics) with two Caesium-137 sources (dose selected based on [92]). Irradiated spores were verified as dead by plating and incubation for 5 days: no growth occurred. The dead spores were microinjected at the same dilution of stock as used for live spores. Irradiated spores still stained well with calcofluor. For intravascular delivery experiments, microinjection of conidia utilised polydimethylsiloxane (PDMS) microstructured surface arrays [93], while imaging was performed following mounting in PDMS imaging devices, as previously described [94].

Leukocyte enumeration
Leukocyte numbers were determined by two techniques. For direct enumeration, manual counting of neutrophil numbers was assisted by the brush tool in Paintbrush 2.1.2 (Soggy Waffles), which records clicks to avoid duplicate counting. Alternatively, "Leukocyte Units" (LUs), a surrogate parameter proportional to leukocyte numbers determined by analysis of digital images, were computed as previously described and validated [61]. LUs incorporate an internally-controlled correction for cell size, and can be independently applied to the signal from fluorescent neutrophils and macrophages. Where appropriate, LUs are called "Neutrophil Units" or "Macrophage Units". In some cases as indicated, leukocytes were enumerated in the tail region distal to the tip of the yolk extension, in order to score cells in a representative part of the whole animal where overlap and anatomical shape did not interfere with accurate scoring. Migrating phagocyte numbers and their phagocytosis of conidia were counted manually in reconstructed 3-dimensional imaged volumes using Imaris v5 (Bitplane).

Gene knockdown by antisense morpholino oligonucleotides
Antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools, LLC (Eugene, OR) (S1 Table). New MOs were demonstrated to target their intended sequence using EGFP reporter constructs engineered to contain target sequences (S10 Fig). MO-csf3r ATG specificity was controlled by MO-csf3r splice ; only MO-csf3r ATG data are presented. MO-il6ra ATG and MO-gp130 ATG served as specificity controls for each other as they targeted separate components of the same heterodimeric receptor complex.

Csf3b overexpression
Capped csf3b mRNA was transcribed from pCS2+ plasmids containing the cDNA [67] linearized by Not1-HF using the mMessage mMACHINE kit followed by RNA cleanup using RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. 5 μL was subjected to RNA electrophoresis for a quality check and stock aliquoted and stored at -70˚C. 500-1000 pg of capped RNA was microinjected directly into the cell of 1-cell embryos. Control embryos received diluent alone.

Mammalian macrophage infection with Talaromyces marneffei
J774 murine macrophages were seeded at a concentration of 1x10 5 conidia/mL into a 6 well microtitre tray containing sterile coverslips and 2 mL of DMEM medium. Macrophages were incubated at 33˚C or 37˚C for 24 hours followed by the addition of 0.1 μg/mL lipopolysaccharide (LPS) and incubation for a further 24 hours. The cells were washed in PBS and 2 mL of complete DMEM medium containing 1x10 6 conidia was added. A control lacking conidia was also performed. Macrophages were incubated for 2 hours at 37˚C to allow conidia to be engulfed, washed once in PBS to remove non-phagocytosed conidia and incubated a further 24 hours at 33˚C or 37˚C. Macrophages were fixed in 4% paraformaldehyde and stained with 1 mg/mL fluorescent brightener 28 (calcofluor) to observe fungal cell walls. Mounted coverslips were examined using differential interference contrast and epifluorescence optics for cell wall staining and imaged on an Olympus IX70 microscope.

Histology and stains
Standard 4% paraformaldehyde-fixed, paraffin-embedded sections were stained by hematoxylin and eosin or Grocott methanamine silver stains by the Walter and Eliza Hall Institute of Medical Research Histology Department. FACS-sorted leukocytes from infected Tg(mpx: EGFP) embryos were collected based on EGFP fluorescence and cytospun preparations stained with Grocott methanamine silver / Nuclear Fast Red.

Microscopy and image processing
Routine brightfield and fluorescence imaging used a Zeiss Lumar V12 stereo dissecting microscope with an AxioCam MRm camera running AxioVision 4.8 software. Images were 1388x1040 pixels. Compound microscopy used an upright Nikon Optiphot-2 microscope with 40x and 100x objectives and a Zeiss AxioCam MRc5 Camera running AxioVision AC (Release 4.5) software. Images were 1292x968 pixels. Confocal microscopy used a Zeiss LSM 5 Live with a Plan-Apochromat 20x, 0.8 NA objective. Software was Zen (Version 4.0). Images were 16-bit 512 x 512 pixels. Z-depth ranged from 0-90 slices with Z-intervals optimized for 1:1:1 X:Y:Z reconstruction. Time intervals were optimized for each experiment and ranged between 30-200 s. Excitatory laser wavelengths were 405 nm for calcofluor, 489 nm for EGFP and 561 nm for mCherry. Emission detection used a BP495-555 filter for calcofluor and EGFP emission and a LP575 filter for mCherry emission.
Image processing was performed using Fiji (ImageJA 1.45b) and Imaris v5 (Bitplane). Colocalization analysis was performed using the Coloc function in Imaris, with thresholds set such that internal colocalization of each channel corresponded to the cell-specific fluorescent signal. Figures were constructed using Adobe CS5 Photoshop and Illustrator.

Statistics
Descriptive and analytical statistics were prepared in Prism 5.0c (GraphPad Software Inc). Unless otherwise stated, data are mean±SEM, with p-values generated from two-tailed unpaired t-tests. (A-C) Images show abrogation of expression from MO-EGFP mRNA (EGFP fused in-frame to an engineered 5' ATG sequence intended to be targeted by the respective antisense morpholino oligonucleotide (MO) being tested) following MO treatment compared to untreated and EGFP mRNA-only controls, demonstrating on-target MO knockdown capability. MO delivery is traced by co-injection with Rhodamine dextran (Rhod).

Supporting information
(D) Physiological specificity of the ATG-targeting csf3r-MO is confirmed by the concordant neutrophil-depletion phenotypes 4 dpi following knockdown of csf3r by either the ATG-MO (1) or splice-targeting morpholino (2). Data are mean+SD, n = 10 embryos/ group, p-values from unpaired two-tailed t-test.