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Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [3H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (P<.05), hyphal elongation (P<.001), colony size, pig-mentation, and 5-HT release (P<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.
The incidence of invasive aspergillosis continues to rise among immunocompromised hosts . Aspergillus fumigatus is most often involved, accounting for >90% of isolates, followed by Aspergillus flavus and Aspergillus terreus .
Host defenses against molds involve innate, humoral, and cell-mediated immunity, and polymorphonuclear leukocytes (PMNs) and macrophages play a dominant role in protecting against asper gillosis, although the contribution of each appears to be site specific . Platelets are the smallest corpuscular components of human blood and, under normal physiological conditions, number from 150 × 109 to 400 × 109 cells/L . They originate in bone marrow and derive from cytoplasmic fragmentation of a giant precursor cell, the megakaryocyte. Platelets are essential to hemostasis and play various roles in antimicrobial host defenses, exhibiting features characteristic of classic cell-mediated immune effector cells . They impair the viability of Staphylococcus aureus and several other microorganisms . The release of platelet microbial peptides (PMPs) or contact-dependent mechanisms could account for this . Moreover, platelets are able to bind to and internalize bacteria and viruses in vitro .
Little is known about the interaction between platelets and Aspergillus species. Human platelets interact directly with A. fumigatus as they become activated and express the glycoprotein CD63 [5, 6]. Moreover, platelets help PMNs attack aspergilli, because optimal activation requires opsonization of hyphae with either fresh or heat-inactivated plasma .
We have observed that serotonin (5-hydroxytryptamine [5-HT]), which is stored in the dense (δ) granules of platelets, acts fungicidally in vitro against Aspergillus species and attenuates fungal virulence . This led us to explore in more detail the interaction between platelets and Aspergillus species.
One clinical isolate each of A. fumigatus and A. terreus were used in this study. Each had been stored in water at room temperature for a maximum of 2 weeks. For long-term storage, both isolates had been frozen. For the experiments, the isolates were grown on Sabouraud’s dextrose agar (SDA; Merck) at 37°C for 4 days. The conidial suspension was harvested by flooding each colony with 2 mL of RPMI 1640 (Sigma-Aldrich) and was adjusted by means of a hemocytometer to a concentration of 1 × 105 conidia/mL, unless otherwise indicated. The size and viability of the inoculum was verified by quantitative colony counts. For hyphal growth, the conidia were incubated for 16 h at 37°C, and ≥90% formed hyphae under these conditions.
Fresh platelets were provided by the Department of Immunology and Blood Transfusion, Innsbruck Medical University. Platelets were collected from healthy blood donors by thrombocytapheresis, using an Amicus cell separator (Baxter).
To investigate the internalization of conidia by platelets, 1 × 108 platelets/mL were labeled with 40 μL of anti-CD42b fluorescein isothiocyanate–conjugated antibody (BD) and incubated for 15 min at room temperature in the dark. CD42b is constitutively expressed on the surfaces of both resting and activated platelets. Conidia were stained with calcofluor white (Polysciences), in accordance with the manufacturer’s instructions. Platelets and conidia were coincubated at an effector to target cell (E:T) ratio of 100:1 and stirred constantly for 15 and 30 min at 37°C. Each preparation was examined using an inverse Axiovert 100 M BP microscope (Carl Zeiss) equipped with a laser scanning module (LSM 510). Z-series optical sections were recorded at 1 μm, using a ×63 oil-immersion lens (numeral aperture, 1.4).
Platelet adherence to Aspergillus conidia was determined by a modification of the spectrophotometric method described by Yeaman et al. . On the basis of results obtained for various microorganisms, ≥15% platelet adherence was considered to constitute highly active platelet adherence . Thrombin-activated (0.03 U/mL; Sigma-Aldrich) platelets served as the control. Microscopic examination was done to confirm conidia-platelet complexes.
The influence of platelets on galactomannan (GM) release was measured by sandwich ELISA (Platelia Aspergillus; Bio-Rad Laboratories), performed in accordance with the manufacturer’s instructions. One hundred microliters each of a 1 × 105 conidia/mL suspension and a 1 × 108 platelets/mL suspension were incubated for 4, 6, 8, and 12 h at 37°C before measurement.
Inhibition of hyphal elongation by platelets was investigated by assessing hyphal elongation of conidia treated or not treated with platelets . One hundred microliters each of the platelet and conidia suspensions (E:T ratio, 100:1) was inoculated into microwell plates (Greiner) and incubated at 37°C. The morphology of the organisms was determined microscopically at 16 h; platelets were lysed with ice-cold water, and fungi were stained with calcofluor white (Polysciences); a micrometer was used to measure their size. The conidial inhibition rate was calculated from the percentage of conidia that did not germinate. Each sample was assessed in triplicate, with 100 fungal elements measured per sample. Untreated and cytochalasin D (cD)-treated fungi served as controls.
To determine the postplatelet effect on aspergilli, conidial suspensions were prepared as described above and coincubated with platelets for 1 and 5 h at 37°C. The postplatelet effect was assessed using a modification of the procedure of Nagl et al. . Briefly, fungi were washed twice with sterile water, centrifuged at 4000 g for 2 min, and resuspended in RPMI 1640. One hundred microliters of undiluted samples and 1:100 and 1:1000 dilutions in water were spread onto SDA by means of a spiral plater (Whitley), incubated at 37°C, and examined visually for growth after 24 h to determine viable counts. The cross-section dimension was analyzed by guide to determine colony size. Colony counts and pigmentation release in untreated and treated samples were determined by visual examination.
Platelet dense-body secretion induced by Aspergillus conidia and hyphae was measured by [3H]–5-HT release according to the method of Des Prez et al. . Untreated and thrombin-treated (0.03 U/mL) platelets served as negative and positive controls, respectively.
For selected experiments, the microfilament inhibitor cD (Sigma-Aldrich) was added to platelets at a final concentration of 20 μmol/L. cD suppresses the formation of contractile micro-tubules and, hence, platelet degranulation.
Each set of experiments was done in triplicate and was repeated at least 3 times on different occasions for each species. Results are expressed as mean ± SD values. Statistical significance was determined by analysis of variance. P < .05 was considered to indicate statistical significance.
A scanning confocal microscopic image of platelet-conidia interaction demonstrated that platelets were unable to internalize conidia but did surround and cover them, as shown in figure 1.
Platelets had the ability to markedly adhere to both Aspergillus isolates. Only minor differences were detected at the concentrations tested. The adherence of untreated and thrombin-activated platelets was 38% ± 3% and 38% ± 4% for A. fumigatus and 40% ± 3% and 36% ± 3% for A. terreus, respectively. No differences were apparent when platelets were activated with thrombin.
Secretion of GM from A. fumigatus and A. terreus was noticeably decreased in the presence of platelets (P < .05) and remained diminished for 12 h, whereas GM release was not affected by cD-treated platelets (figure 2).
Fungal germination was significantly decreased by platelet treatment (P < .001), with 57% and 52% of conidia of A. fumigatus and A. terreus germinating, respectively, compared with 90% and 83% of untreated controls. Hyphal elongation of A. fumigatus (188 ± 137 μm) and A. terreus (152 ± 132 μm) were significantly decreased by platelet treatment, compared with that for untreated hyphae of A. fumigatus (556 ± 150 μm) and A. terreus (581 ± 230 μm), after 16 h of incubation. Platelets that were treated with the microfilament inhibitor cD did not reduce conidial germination; 90% of A. fumigatus and 92% of A. terreus conidia still germinated. In addition, hyphal elongation of A. fumigatus (433 ± 160 μm) and A. terreus (468 ± 166 μm) were not affected by cD-treated platelets. cD tested alone with Aspergillus species had no effect on germination and hyphal elongation.
Coincubation of conidia with platelets for 1 h and 5 h resulted in a significantly smaller colony size for A. fumigatus (5.2 ± 1.2 and 5.0 ± 0.8 mm) and A. terreus (4.1 ± 1.0 and 3.6 ± 0.9 mm) (P < .05), compared with that for untreated A. fumigatus (7.4 ± 2.1 mm) and A. terreus (6.1 ± 1.2 mm), after 24 h of incubation. In addition, a lack of pigmentation release was observed for A. terreus by visual examination.
Colony counts were 1 × 103 and 1 × 102 colonies/mL for A. fumigatus and A. terreus, respectively. cD-treated platelets had no effect on A. fumigatus and A. terreus.
[3H]–5-HT was released by platelets in response to Aspergillus species and thrombin. Secretion was observed after 30 min, without significant time-dependent differences. Aspergillus hyphae stimulated 5-HT release in a manner similar to thrombin, a strong platelet activator, whereas cD-treated platelets released significantly less 5-HT (P < .001) (data not shown).
This study has demonstrated that platelets interact with Aspergillus species by adhering to conidia, although they are unable to internalize them. Platelet treatment significantly decreased fungal GM release, hyphal elongation, and colony size. Granule release in platelets was meditated by conidia and hyphae of A. fumigatus and A. terreus. By contrast, cD-treated platelets were incapable of degranulation and lacked antifungal activity.
Platelets (3 μm)  were able to surround the conidia of Aspergillus species but could not ingest them, possibly due to the large size of the fungal elements (2–4 μm) , given that platelets have been shown to fail to ingest large latex spherules (3.13 μm), although not particles of smaller sizes . Nonetheless, platelets may mount an attack on the fungus by surrounding and covering conidia and hyphae (figure 1).
Adherence was similar for both species tested, suggesting a generic characteristic that contrasts with what is known about Candida species . Fibrinogen binds to fungal cell surfaces and has been postulated to play a significant role in platelet attachment to Candida albicans . This might also occur with Aspergillus species, given that conidia also interact with fibrinogen and laminin . Adherence of platelets to aspergilli could be the critical event in inducing the activation of platelets .
The polysaccharide GM is an important structural component of the cell wall of Aspergillus species and is released by growing and vital hyphae . Cell wall biosynthesis is a key process in fungal growth , and the results of our study indicate that platelets can alter the quantity of released GM. The ability of Aspergillus organisms to undergo morphological changes from conidia to filaments is necessary for invasiveness, because only hyphae are seen in tissues . The reduced germination and hyphal elongation of Aspergillus organisms when exposed to platelets indicates that they may be able to attenuate the fungal virulence of A. fumigatus and A. terreus.
Several platelet-secretion products are known to exhibit anti-bacterial activity , and PMPs could exert antifungal effects . The release of 5-HT could support this, because it is known that it possesses antifungal properties. However, a selective effect of 5-HT on hyphae is unlikely, because the concentrations necessary for fungal killing exceed physiological levels. Nevertheless, platelets released 5-HT immediately on contact with fungi. A prolonged incubation time of 60 min did not increase the release, as has been shown for Histoplasma capsulatum .
Thrombocytopenia is associated with a greater risk of invasive fungal disease after liver transplantation , and it has been observed that patients with invasive fungal diseases had a significantly longer duration of thrombocytopenia than did those without infection (P < .004) . Our in vitro findings suggest an explanation for these clinical observations, one that warrants further investigation.
In conclusion, we have shown that platelets are able to impair and reduce fungal viability and virulence in vitro, probably by means of granule-dependent mechanisms. This suggests that platelets play a key role in antifungal host defenses.
Financial support: Austrian Science Foundation (grant FWF-P17484-B05).
Potential conflicts of interest: none reported.
Presented in part: 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 16 –19 December 2005 (poster and abstract M-918); 2nd Advances against Aspergillosis Conference, Athens, 22–25 February 2006 (poster and abstract P-151).