Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function

Microbes and Infection 4 (2002) 1427–1438 www.elsevier.com/locate/micinf

Original article

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function Lesley Wright a,*, William Bubb b, John Davidson a,e, Rosemary Santangelo a, Mark Krockenberger c, Uwe Himmelreich a,d, Tania Sorrell a a

Department of Infectious Diseases, Center for Infectious Diseases and Microbiology, Level 3, ICPMR Building, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia b Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia c Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia d Department of Magnetic Resonance in Medicine, Institute for Magnetic Resonance Research, University of Sydney, Sydney, NSW 2006, Australia e Department of Biological Sciences, University of Western Sydney, Nepean, Kingswood, NSW 2747, Australia Received 11 July 2002; accepted 10 September 2002

Abstract Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4–5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites—acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and c-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cryptococci; Neutrophils; Metabolites; Function; Magnetic; Resonance; Spectroscopy

1. Introduction Cryptococcus neoformans var. gattii (CNVG) differs from C. neoformans var. neoformans (CNVN) in its predilection to cause disease in immunocompetent hosts and in its more frequent association with localized lesions (cryptococcomas) in the lung and brain [1]. In an early study, reported in abstract form, it was noted that neutrophil infiltrates were more prominent in the lungs of mice infected with C. neoformans serotypes A or D, than those infected with serotypes B

* Corresponding author. Tel.: +612-98457367; fax: +612-98915317. E-mail address: [email protected] (L. Wright) © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 0 0 2 4 - 2

or C [2]. In serotypes A and D, the time-course of pulmonary infection is characterized by an early neutrophil response, with phagocytosis of the cryptococci noted at day 7 [3]. Phagocytosis and killing of cryptococci by neutrophils is diminished in the presence of a large polysaccharide capsule, but enhanced by the binding of complement-derived opsonins, such as C3, to the capsule [4]. The capsular serotype influences the rate of binding of C3 to the polysaccharide capsule, e.g. binding to serotypes B and C is slower than to serotypes A and D [5], and in serotypes B and C the C3bBb C3 convertase (involved in the alternate pathway of complement activation) is inefficiently assembled on the capsule surface [6]. These findings suggest that CNVG might be less

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efficiently phagocytosed by neutrophils and other phagocytes than CNVN. Furthermore, filtrates of supernatants (CneF) from cultures of CNVN, but not CNVG, contain chemotactic and chemokinetic activity and the filtrates from CNVG inhibit neutrophil chemotaxis [7]. Capsular glucuronoxylomannan (GXM) was identified as the chemotactic principle in CneF from CNVN [7]. Others have reported that intraperitoneal injection of very high concentrations of CNVG-derived supernatants reduced the extent of inflammation in a mouse model of staphylococcal arthritis [8]. In addition to the polysaccharide capsule, other components secreted by C. neoformans, which might affect neutrophil function, have been identified [9]. We utilized the technique of magnetic resonance spectroscopy (MRS) to identify non-enzymic, extracellular cryptococcal products of CNVN with the potential to influence the function of mammalian cells. The most abundant of these were ethanol, acetic acid, mannitol, glycerol, glucitol, erythritol, c-aminobutyric acid (GABA), choline and ethanolamine derivatives, nucleosides, and amino acids (including arginine) [9]. Acetic acid, mannitol, glycerophosphorylcholine, ethanol and small amounts of GABA were identified by MRS in cryptococcomas harvested from the brains of experimental rats infected with CNVG [10]. It was proposed by Dong and Murphy [7] that lack of an early, predominantly neutrophil, response to CNVG results in reduced pulmonary clearance, thereby favoring development of cryptococcomas. This hypothesis has not been tested. In the present study we performed early time-course studies of CNVG pulmonary infection in a rat model, compared the profile of MRS-visible extracellular metabolites of CNVG and CNVN and tested the effect of heat-inactivated cryptococcal supernatants and selected components identified in them, on neutrophil viability and function.

2. Materials and methods 2.1. Cryptococcal strains BL-1, a serotype A isolate from human lung, and TCSSC1 a serotype B, pathogenic isolate originally cultured from a eucalyptus tree (Eucalyptus tereticornis) at Mt. Annan, NSW, Australia, were obtained from the Westmead Hospital cryptococcal culture collection. 2.2. Rat model of pulmonary cryptococcosis due to CNVG CNVG strain TCS-SC1 maintained on Sabouraud’s dextrose agar (SDA), was inoculated into yeast nitrogen broth and cultured at 28 °C to early stationary phase (ca. 18 h). Cells were centrifuged, washed in phosphate-buffered saline (PBS) and adjusted to a concentration of 108 CFU/ml. Female Fischer 344 rats (Animal Research Council, Perth,

Australia), weighing 150–250 g, were anesthetized by inhalation of 4% halothane in 100% oxygen prior to intraperitoneal injection of 11.6 mg/kg body weight of ketamine hydrochloride (Apex Laboratories, Sydney, Australia) and 1.2 mg/kg of xylazine hydrochloride (Apex Laboratories). Lignocaine spray (4%) was administered to the pharynx to inhibit coughing and the vocal chords were visualized through a dog otoscope to guide insertion of a long, bent, blunt-ended 18-gauge steel needle, containing 0.1 ml of the cryptococcal suspension, into the trachea. This suspension was delivered into the trachea, followed by a 0.6 ml burst of air, to disperse the inoculum into the small airways and the alveoli. Post-operatively, rats received a single intraperitoneal injection of Clavulox (140 mg/ml amoxycillin and 35 mg/ml clavulanic acid (Pfizer Pharmaceuticals, Australia) to prevent secondary bacterial infection. Control rats received 0.1 ml PBS instead of cryptococcal cell suspension. Animals were given oxygen and allowed to recover under a heat lamp. At specified times, rats were euthanazed by inhalation of CO2. The lungs, heart and mediastinum were removed in a block from the chest cavity. The lungs and trachea were dissected out, fixed in formalin, embedded in paraffin, sections prepared and stained with hematoxylin and eosin (H&E) or with Mayer’s mucicarmine (to highlight cryptococcal capsules) prior to microscopy. Whole lungs from two animals per time point were also utilized to determine cryptococcal load. Tissue was weighed, homogenized in sterile normal saline, serial dilutions made, plated on to SDA, incubated at room temperature for 3–5 days and colony counts determined per gram of tissue. 2.3. Measurement of pH in rat cryptococcomas Two male Wistar rats were anesthetized as above, and inoculated intracranially with CNVG strains McBride C63 and C64 by the method outlined in [10]. Ten days later, these rats and one uninoculated control rat were anesthetized, the brains rapidly excised at the point of death, and the cryptococcal lesions located. The pH was measured in the center and near the periphery of the lesions, and in control brain tissue following insertion of a microcombination pH electrode (tip diameter 660 µ, World Precision Instruments, Inc., USA) within 2–3 min of death. All animal experimentation was carried out according to the Australian National Health and Medical Research Council guidelines and with approval from the University of Sydney Ethics Committee. 2.4. Elimination of endotoxin Glassware was baked at 200 °C before use, endotoxin-free reagents were purchased where possible, and all other reagents, including purchased chemicals and supernatants, were ascertained to be endotoxin-negative using the E-toxate kit (Sigma).

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2.5. Preparation of heat-inactivated cryptococcal supernatants Cryptococcal supernatants were prepared by the method of Chen et al. [11,12] from isolates grown to confluence on SDA for 72 h at 30 °C. Briefly, colonies were scraped from the plates, washed in saline, and resuspended in a volume equal to that of the cell pellet of harvesting buffer (10 mM imidazole, 2 mM CaCl2, 2 mM MgCl2, 56 mM D-glucose, in 0.9% NaCl, pH 5.5). This buffer was used so that secreted metabolites could easily be identified by MRS, without interference from media components. It was not toxic to cryptococci; indeed, there was an increase of 10% in the number of colony-forming units after 20–24 h of incubation at 37 °C. Cells were removed from supernatants by centrifugation twice at 12 000 g for 15 min. Supernatants were assayed for protein content using the BIO-RAD assay kit, BIO-RAD, Ryde, NSW, Australia. Supernatants from strains BL-I and TCS-SC1 (termed vns and vng, respectively), had similar protein contents, but buffer was added, if necessary, to ensure that the protein concentrations were equal. Heat-labile components in the cell-free supernatants were inactivated at 80 °C for 10 min and the pH was measured, then adjusted to 5.5 or 7.0 with endotoxin-free NaOH prior to storage at – 70 °C. The concentration of cryptococcal polysaccharide (GXM) was estimated using the standard cryptococcal latex agglutination test (CALAS, Meridian Diagnostics Inc., Cincinnati, OH, USA). 2.6. MRS samples Supernatants were obtained from the 1.0 ml packed volume of cells from each cryptococcal strain. Assignments of metabolites were made from spectra of cryptococcal supernatants, which had been concentrated four-fold by lyophilization of 2 ml aliquots and re-dissolution in 500 µl of 2 H2O. Volatile metabolites were identified in, and all concentrations were estimated from, 550 µl samples of untreated supernatants. For the latter samples, a coaxial capillary containing 1 mM sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate (TSP-d4 sodium salt, from Commissariat a l’Energie Atomique, Gif-sur-Yvette, cedex, France) in 2H2O was used as field-frequency lock.

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programs were used without modification. Concentrations were assessed, where possible, from fully-relaxed 1H-MR spectra and otherwise from integration of cross-peak volumes in heteronuclear single quantum coherence spectra as described and discussed by Bubb et al. [9]. 2.8. 13C MRS time-course experiments Cryptococci were incubated in harvesting buffer containing 5 mM [2-13C]glucose. Supernatants of the cell suspensions were studied by 13C MR spectroscopy after 0, 2, 4, and 16 h of incubation at 37 °C. An aliquot (10% of final volume) of 2H2O was added to the supernatants prior to the MRS experiments. Spectra were obtained on a Bruker Avance-360 NMR spectrometer operating at a frequency of 90.55 MHz, with 512 transients averaged into 64 k data points. A spectral width of 20 000 Hz and a relaxation delay of 5 s were used. Inverse gated decoupling was applied to minimize both sample heating and enhancement of resonance intensities of 13 C nuclei due to the nuclear Overhauser effect. Chemical shifts are expressed relative to the a-glucose C-2 carbon resonance at 72.7 ppm. Metabolite resonances were assigned from the 13C chemical shift data obtained in the 2-D MRS studies. 2.9. 1-D 31P MRS experiments These were obtained using a Bruker Avance-400 NMR spectrometer at a frequency of 161.98 MHz, with 1024 transients averaged into 64 k data points. Samples (packed cryptococcal cells or untreated, non-lyophilized supernatants containing approximately 10% 2H2O) were placed in 10 mm MRS tubes (Wilmad Glass Co. Inc., Buena, NJ, USA). Spectra were acquired immediately after placing the sample in the magnet (5 min time point) and after 1, 2, and 4 h at 37 °C. The spectral width was 8000 Hz, the relaxation delay was 5 s, and inverse gated decoupling was used. 31P-MR spectra were also acquired from supernatants at zero time, 2, 4, and 16 h. An aliquot of 2H2O (final concentration 10%) was added to the supernatants prior to the MRS experiments. Chemical shifts were referenced to external 2 mM methylphosphonate at 23.0 ppm. Metabolites were identified by comparison of chemical shifts with published data.

2.7. MR spectroscopy 2.10. Isolation of neutrophils Metabolites were identified by determination of 1H and 13 C chemical shifts and their associated correlations using the comprehensive MRS methods previously described [9] and comparison with published data. Spectra were acquired on a Bruker Avance-600 NMR spectrometer with a tripleresonance inverse-detection xyz-gradient probe at a 1H frequency of 600.14 MHz, with the variable temperature unit set at 25 °C. Chemical shifts are expressed relative to the anomeric resonance of a-glucose (1H, d 5.223 ppm; 13C, d 92.9 ppm). Standard Bruker (XWIN-NMR version 2.5) pulse

Neutrophils were prepared from blood of healthy Blood Bank volunteers as previously described [13]. Leukocytes were sedimented with 3.5% dextran and neutrophils were separated from other cell types by centrifugation on Ficoll– Paque. Residual erythrocytes were removed by hypotonic lysis and neutrophils resuspended in calcium and magnesium-free phosphate buffered saline [PBS(–)]. Cell purity (> 90%) was monitored by Coulter Counter (Coulter, Hialeah, FL, USA).

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2.11. Neutrophil viability This was determined by flow cytometry as described previously [9], using Annexin V-Fluos (Boehringer–Mannheim Australia, Castle Hill, NSW, Australia) and propidium iodide (Sigma, St. Louis, MO, USA). 2.12. Incubation of neutrophils with cryptococcal supernatants or metabolites Neutrophils (107 cells per ml) were pre-incubated for 2 h in a water bath at 37 °C with gentle agitation in either heat-inactivated cryptococcal supernatants or harvesting buffer alone at pH 5.5 or 7.0. Pure chemicals were also incubated with neutrophils under these conditions.

added to six bare filters on the same plate. The Transwells were then incubated at 37 °C for 1 h in a CO2 incubator. Non-adhering neutrophils were removed by extensive washing of the filters, and the radioactivity of these cells was counted. Neutrophils which had migrated through to the bottom compartment were also counted for radioactivity. The filters were treated with chloroform and methanol to dissolve out the radio-labeled lipids from strongly adherent/migrated neutrophils. The solvents containing lipids were transferred to scintillation vials, dried under nitrogen, and the radioactivity was counted. The percentage of the total counts recovered in the filters and the lower Transwell chamber was taken as the percentage of migrated neutrophils. Triplicate filters/wells were used for all conditions.

2.13. Superoxide production by neutrophils

3. Results

Superoxide production was measured by the method of Babior et al. [14], and modified for use in a microtitre spectrophotometric assay as the superoxide-dismutase inhibitable reduction of ferricytochrome C (horse-heart, Sigma). Absorbances were read at 550 nm, with 0.3 cm path length. The extinction coefficient used for calculations was 29.5 mM/cm. Neutrophils were pre-treated as described above, washed, suspended in Hanks’ balanced salt solution (HBSS) and stimulated with either 4b-phorbol 12-myristate acetate (PMA) or formylmethionyl leucyl phenylalanine (fMLP) at 1.6 and 1 µM final concentration, diluted from ethanol or DMSO stock solutions, respectively. Controls contained HBSS and equivalent amounts of these solvents. Assays were performed in triplicate.

3.1. Histopathology of pulmonary infection due to CNVG

2.14. Migration assays The human lung epithelial cell line A549 (obtained from the ATTC collection, USA) was used for these experiments and cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal calf serum. Cells (200 µl) were inoculated onto six 3.0 µm pore-size filters in a Transwell plate containing 12 6.5-mm diameter wells (Corning Inc., Corning, NY, USA). The remaining six filters were left bare. DMEM (1 ml) was added to the compartment below the cells, which were grown to confluence at 37 °C in 5% CO2. Confluence was confirmed by phase-contrast microscopy. Neutrophils were labeled by pre-incubation in HBSS with 5 µCi of carrier-free [9,10(n)-3H] oleic acid for 1 h at 37 °C with gentle shaking [15]. After washing in PBS(–) containing 1 mg/ml BSA, they were incubated in either cryptococcal supernatants or harvesting buffer (untreated controls) for 2 h, washed in HBSS and resuspended in HBSS at a concentration of 5 × 105 cells in 100 µl. Medium was replaced in the lower compartments of the Transwell plates with either HBSS alone or 1 µM fMLP in HBSS. The medium above the A549 cells was replaced with 100 µl of neutrophil suspension in HBSS, and 100 µl of neutrophil suspension were also

The time course of rat pulmonary infection by CNVG is shown in Fig. 1. An example of naturally acquired infection in a koala has been included for comparison. In the rat model, a mixed inflammatory infiltrate comprised of approximately 50% neutrophils, with some eosinophils and macrophages, was noted on day 1. Cryptococci were few in number and their capsules remained small. Several had been engulfed by alveolar macrophages. By day 4, neutrophils were less prominent, comprising about 5–10% of inflammatory cells. Approximately 10% were eosinophils and the remainder, macrophages. Again, few cryptococci were visible. Extracellular cryptococci had developed large capsules, in contrast to those within macrophages. Small granulomas were now apparent. By day 12, the neutrophil response had largely abated, and cryptococci, which showed evidence of budding, were present in large airways as well as alveoli. By day 36, large aggregates of cryptococci were found in large airways and alveolar spaces. Eosinophils were prominent (up to 20%). At no time were cryptococci seen within neutrophils. 3.2. Composition of cryptococcal supernatants The pH values in vns and vgs were 3.5–3.8 and 4.0–4.5, respectively. Capsular antigen (GXM) was present in each in a concentration of approximately 500 µg/ml. The concentrations of MR-visible metabolites in vns and vgs are summarized in Table 1. Acetate and ethanol were the dominant components, followed by polyols, amino acids, nucleosides, ethanolamine and choline derivatives, and organic acids. It is notable that the most abundant metabolites in vgs are qualitatively similar to, but generally of a lower concentration to those in vns, especially acetate and ethanol. The higher acetic acid content of the vns is reflected in its lower pH (see above). Two metabolites in vgs were not apparent in vns, namely, dihydroxyacetone (CH2OH, d 1H, 4.42, d13C, 65.7; CO, d 13C, 212.8), and acetoin (CH3, d 1H, 1.37, d 13C, 19.1;

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Fig. 1. The time course of rat pulmonary infection by CNVG. In all cases the bar = 17 µm, except for (F), which is 170 µm. (A) Rat model of pulmonary cryptococcosis, day 1. Stain H&E, magnification × 200. The cryptococcal cells (arrow) are hard to visualize. The fungal cells are small, with small capsules and are surrounded by an acute host response comprised mainly of neutrophils (PMN, arrow). (B) Rat model of cryptococcosis, day 1. Stain, Mayer’s mucicarmine, magnification × 200. Cryptococci (arrow) are now stained red. (C) Rat model of pulmonary cryptococcosis, day 4. Stain, Mayer’s mucicarmine, magnification × 200. Cryptococci (arrow) surrounded by a host inflammatory response now dominated by mononuclear cells, presumably derived from pulmonary macrophages. The cryptococci are small with mostly very little capsular material. In some parts of the lung there were variable numbers of scattered and local aggregations of neutrophils (not shown). (D) Rat model of pulmonary cryptococcosis, day 12. Stain, Mayer’s mucicarmine, magnification × 200. Note the large cryptococcal capsule (stained red). There are few neutrophils and those inflammatory cells which are present, are mainly mononuclear. (E) Rat model of pulmonary cryptococcosis, day 36. Stain, Mayer’s mucicarmine, magnification × 200. This large CNVG cell is budding and surrounded by a granulomatous, inflammatory response dominated by epithelioid cells. Few neutrophils are present throughout the lung. (F) Rat model of pulmonary cryptococcosis, day 36. Stain, Mayer’s mucicarmine, magnification × 20. This is a low-power view of a cryptococcoma. The host response is concentrated around the periphery of the lesion. (G) Rat model of pulmonary cryptococcosis, day 36. Stain, H&E, magnification × 200. Giant cell formation (arrow), in association with cryptococci. (H) Naturally occurring pulmonary cryptococcosis in a koala. Stain, H&E, magnification × 200. Many neutrophils surround a large encapsulated CNVN cell. This koala had chronic nasal cryptococcosis with later central nervous system involvement, and recent dissemination to the lungs via the airways. No macroscopic pulmonary lesions were seen and very few fungal cells were visualized microscopically.

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Table 1 Comparison of metabolite production from C. neoformans var. neoformans (BL-1) and C. neoformans var. gattii (TCS SC-1)a Moleculeb

BL-1

TCS-SC1

Leucine Isoleucine Valine Ethanol Lactate Threonine Alanine Acetate Acetoin GABA Succinate Methionine Aspartate Lysine Choline GPC Arginine Glycinebetaine GPE Glycerol Glycine Erythritol Glucitol Mannitol Serine Dihydroxyacetone a, a-trehalose Cytidine Tyrosine Phenylalanine Uridine Guanosine Adenosine Acetaldehyde

1 1 2 62 2 1 5 172 nd 4 1 0.3 0.9 < 10 0.3 2 2 3 0.7 23 1 7 4 8 1 nd V 0.2 0.5 1 0.8 0.6 2 0.1

0.4 0.3 0.4 28 0.6 0.5 3 92 0.2 1 0.5 0.2 T <9 0.2 0.8 1 1 1 15 0.5 1 2 2 T 0.2 0.4 0.1 0.2 0.6 0.3 0.2 0.5 T

a Concentrations (mM) were generally estimated from integration of fully-relaxed 1H-NMR spectra after subtracting independently measured contributions where necessary. Details for the estimation of concentrations of specific metabolites for which the general approach could not be used are provided in Bubb et al. [9]. Samples were freshly prepared—i.e. not concentrated by freeze-drying. The buffer was harvesting buffer pH 5.5 (see Methods). nd, not detected; T, trace amount only, although detected in both 1-D and 2-D spectra; V, variable. b Assignments were made after consideration of all possible contributions to the relevant signal intensity, according to Bubb et al. [9].

CHOH, d1H, 4.43, d 13C, 73.9; CH3, d 1H, 2.23, d 13C, 25.7; CO, d13C, 216.2). The time course for the production of some of the secreted metabolites was studied using 1-D 13C-MR spectroscopy. In both cryptococcal strains, exogenous [2-13C] glucose was rapidly transformed into acetate and ethanol (detectable in the supernatants after 2 h, Fig. 2). Glycerol was also detectable after 2 h. The concentration of metabolites in vgs was again found to be lower relative to vns. The rate of glucose consumption was higher in vns (ca. 0.38 mmol/h per l of packed cell suspension) relative to vgs (ca. 0.31 mmol/h per l

of packed cell suspension) under our experimental conditions (Fig. 2). 3.3. 1-D 31P MR spectroscopy This revealed that in addition to glycerophosphorylcholine and glycerophosphorylethanolamine, large amounts of inorganic phosphate accumulated rapidly in supernatants of both cryptococcal varieties. Time courses in both were similar, therefore, only vgs is illustrated in Fig. 3A–D and a–d. 31 P MR spectra of cryptococcal cells incubated in harvesting buffer at pH 5.5 for 5 min, 1, 2, and 4 h revealed the presence of polyphosphate (Fig. 3A–D). Polyphosphate concentrations decreased with time, in inverse proportion to the accumulation of inorganic phosphate, which was presumably then released to the supernatant (Fig. 3a–d). Degradation of intracellular nucleoside triphosphates, such as ATP, was also noted. 3.4. Effects of pH and cryptococcal supernatants on neutrophil viability Since we observed that substantial amounts of acetic acid were produced by cryptococci in vitro (see above, and [9]) and in a rat model of cerebral cryptococcoma [10], we tested the pH of the latter from two rat brains. The pH inside the cryptococcomas was 5.4–5.6 in both rats. In the rat more severely affected (meningitis), even the tissues well outside the lesion were acidic (pH 6.3–6.7). In the rat with a small lesion only, the tissue far from the unaffected area was pH 7.5. In human tissues, acidic conditions are common in sites of inflammation to which neutrophils migrate [16], with pH values of 5.7–7.2 being recorded in pus from bacterial infections [17]. We were able to confirm by MR spectroscopy that acetic acid was present in brain tissues from rats with cryptococcomas, but not in the tissue from the uninfected control rat (results not shown). Based on these observations, we chose to observe the effects of pre-incubation of neutrophils in buffer at pH 5.5 and 7.0, or vns and vgs adjusted to pH 5.5 and 7.0 on neutrophil viability and function. The results for viability are summarized in Table 2A, B. As determined previously [18], there was no difference between neutrophils incubated in buffer alone at pH 5.5 or 7.0; that is, no decrease in viability was observed by lowering the pH to 5.5. Different batches of neutrophils with different initial viabilities (not shown) were used in Table 2A, B. These were not significantly different from their corresponding untreated controls, which were incubated in buffer alone. However, both supernatants, but especially vns, produced substantial necrosis at pH 5.5, based on increased permeability to propidium iodide, though there was no evidence of neutrophil clumping or cell lysis. Vns also caused a small increase in necrotic and apoptotic cells at pH 7, in agreement with our previous experiments [18].

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Fig. 2. 13C-NMR spectra of supernatants from C. neoformans cultures containing 5 mM [2-13C]glucose. Supernatants were harvested at zero time (A), 2 h (B), 4 h (C), and 16 h (D). Spectra represent vns (left column, I) and vgs (right column, II). Abbreviations: a-glc, a-glucose; b-glc, b-glucose.

3.5. Effects of pH and supernatants on neutrophil function 3.5.1. Superoxide production—effects of low pH Despite there being no decrease in the viability of neutrophils pre-incubated in buffer alone at pH 5.5, there was a

dramatic decrease in superoxide production after preincubation at pH 5.5 compared with pH 7.0 (Table 3). This difference occurred when superoxide production was measured in HBSS (controls), and was even more pronounced after stimulation in HBSS with PMA or fMLP (Table 3).

Fig. 3. 31P-NMR spectra of packed cell suspensions of CNVG obtained 5 min (A), 1 h (B), 2 h (C), and 4 h (D) after suspension in harvesting buffer at 37 °C. Spectra were also acquired from supernatants prepared from cell suspensions at zero time (a), 2 h (b), 4 h (c), and 16 h (d) at 37 °C. NMR experiments were performed after the addition of 2H2O, as described in Materials and methods. A total concentration of 2 mM methylphosphonate (MeP) was added to the supernatants. Abbreviations: Pi, inorganic phosphate; PP, polyphosphates; GP, glycerophosphates; NTP, nucleoside triphosphates.

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Table 2 The effects of cryptococcal supernatants on the viability of human neutrophils Treatment

Percentage of cellsa

(A) pH 5.5 Untreated vns vgs

Normal 86.2 ± 1.1 11.7 ± 1.7b 32.9 ± 2.2b,c

Apoptotic 1.5 ± 0.3 0.5 ± 0.25 1.0 ± 0.7

Necrotic 12.7 ± 1.1 87.8 ± 1.9b 66.1 ± 1.8b,c

(B) pH 7.0 Untreated vns vgs

92.8 ± 1.0 88.5 ± 0.1 92.9 ± 0.5

3.3 ± 0.7 7.3 ± 0.6 4.2 ± 0.5

2.8 ± 0.6 4.8 ± 0.2 2.7 ± 0.1

a

Values are means ± S.E.M. of three to seven experiments for pH 5.5 and two to four experiments for pH 7.0. b Significantly different from untreated controls, which were incubated in buffer only. P < 0.01 by the paired two-tailed t-test. c Significantly different from vns, P < 0.01 by the paired two-tailed t-test.

3.5.2. Superoxide production—effects of cryptococcal supernatants The trend to lower superoxide production was exacerbated after pre-incubation of neutrophils in the cryptococcal supernatants at pH 5.5, probably due to the decreased viability of the cells (Table 4A). There was virtually no response to stimulation by PMA or fMLP, although the differences in superoxide production in the cells pre-incubated with vns or vgs from the untreated controls did not reach significance. At pH 7.0, neutrophils incubated in cryptococcal supernatants produced significantly larger amounts of superoxide in response to stimulation (Table 4B) compared with those at pH 5.5 (Table 4A). Incubation with vns produced a significantly greater amount of superoxide in response to PMA compared with the untreated cells, whereas those incubated with vgs produced significantly less superoxide (Table 4B). Incubation of neutrophils with either of the cryptococcal supernatants produced an apparently similar increase in the neutrophil response to fMLP when compared with the untreated cells, but this did not reach significance (Table 4B). 3.5.3. Effects on adhesion/migration We studied the effect of pre-incubating neutrophils with supernatants adjusted to pH 5.5 on their ability to migrate towards the chemoattractant, fMLP, in the presence of a Table 3 Effects of pH on superoxide production by human neutrophils pre-incubated in imidazole buffer

Stimulus

Superoxide production (nmol per 107 cells per 30 min)a pH 7.0 pH 5.5

Control (HBSS only) + PMA + fMLP

19.1 ± 1.8b 95.2 ± 4.5b,c 40.1 ± 3.6b,c

a

8.1 ± 1.1 18.4 ± 2.2c 10.1 ± 0.5c

Results are expressed as means ± S.E.M. of 10 experiments. Significant difference from pH 5.5, P < 0.001 by the unpaired twotailed t-test. c Significant difference from controls, P < 0.001, or P = 0.05 (pH 5.5, + fMLP), by the unpaired two-tailed t-test. b

Table 4 Response to stimulation by PMA and fMLP of neutrophils after preincubation with imidazole buffer or cryptococcal supernatants at pH 5.5 or 7.0 (A) pH 5.5 + PMA + fMLP (B) pH 7.0 + PMA + fMLP

Superoxide production (nmol per 107 cells per 30 min)a Untreated (buffer only) vns vgs 9.0 ± 3.6 2.0 ± 0.9 70.5 ± 3.8b 25.4 ± 4.7b

1.4 ± 0.8 1.5 ± 0.8

2.4 ± 1.1 1.6 ± 0.6

81.2 ± 1.9b,c 59.3 ± 1.7b,c 34.2 ± 4.6b 36.8 ± 8.4b

a Results are expressed as means ± S.E.M. of four experiments. The corresponding control values of superoxide production from unstimulated neutrophils incubated in HBSS alone (see Table 3) have been subtracted from all numbers in A and B. b Significant difference from pH 5.5, P < 0.0001 by the unpaired twotailed t-test. c Significant difference from untreated cells, P < 0.05 by the paired two-tailed t-test.

monolayer of human lung epithelial cells (A549) and through bare filters. This methodology measures both migrated and strongly adhered cells. Unstimulated migration/adhesion to bare filters was significantly reduced after pre-incubation with either vns or vgs at pH 5.5 (Fig. 4A, C). There was no chemotaxis of control neutrophils (incubated in pH 5.5 buffer, alone) towards fMLP, or of neutrophils preincubated with the cryptococcal supernatants (Fig. 4A, C). After preincubation in pH 7.0 buffer, normal chemotaxis towards fMLP was observed (data not shown). As expected, migration/adhesion of control neutrophils in the presence of A549 cells was less than through bare filters (compare Fig. 4A, B and C, D). Again, there was no chemotactic response to fMLP of either the control cells or the supernatant-treated cells, both of which had been preincubated at pH 5.5. Neutrophils pre-treated with vns, however, exhibited increased migration/adhesion through A459 cell monolayers compared with the untreated controls (Fig. 4B), whereas there was no such increase with vgstreated cells (Fig. 4D). 3.6. Effects of cryptococcal metabolites on neutrophils Relatively abundant cryptococcal metabolites with the potential for pathogenicity were tested for their effects on neutrophil viability and superoxide production. Cells were incubated with metabolites at pH 5.5 and 7.0 using the concentrations based on those measured for vns (see Table 1). Glycerol and glycerophosphorylcholine had no effect. A combination of polyols (mannitol, glycerol, glucitol, erythritol) stimulated superoxide production at pH 7.0 directly and in response to fMLP, but decreased the neutrophil superoxide response to PMA (Table 5). There was no significant effect of polyols on neutrophil superoxide production at pH 5.5 (data not shown) and they induced neither significant necrosis nor apoptosis at pH 5.5 or 7.0 (data not shown). Two components found only in vgs, dihydroxyacetone and acetoin, like the polyols, induced more apoptosis at both

L. Wright et al. / Microbes and Infection 4 (2002) 1427–1438

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Fig. 4 . The effects on adhesion/migration of human neutrophils of incubation with cryptococcal supernatants. Empty bars represent migration of neutrophils through bare filters (A and C) and solid bars represent migration of neutrophils through a monolayer of A549 cells (B and D). Neutrophils were radiolabeled as described in Materials and methods, then pre-incubated with harvesting buffer (untreated controls) or vns or vgs for 2 h at 37 °C. The neutrophils were then allowed to migrate towards 1 µM fMLP or HBSS in the bottom well of the Transwells for 1 h at 37 °C as described in Materials and methods. The results are expressed as the means and S.D. of six separate experiments in duplicate or triplicate. a, Significantly different from bare filter untreated controls. b, Significantly different from bare filter untreated controls + fMLP. c, d, Significantly different from A549 untreated controls or A549 untreated controls + fMLP, respectively. e , Significantly different from bare filter, vgs-treated cells. Comparisons carried out using the paired two-tailed t-test; P < 0.05.

pH 5.5 and 7.0, but this did not reach statistical significance. Dihydroxyacetone directly stimulated neutrophil superoxide production at pH 5.5 (7.9 ± 2.3, untreated, vs. 11.2 ± 1.7, treated, expressed as nmol per 107 cells per 30 min, P = 0.0395, n = 4, data not shown). At pH 7.0, there was a small but significant increase in the neutrophil superoxide response to PMA and a decrease in response to fMLP (Table 6).

None of GABA, ATP or adenosine affected neutrophil viability or superoxide production at pH 7.0. GABA enhanced the neutrophil superoxide response to PMA at pH 5.5 (Table 7) corresponding with a very small, but consistent, increase in viability (about 5%, data not shown).

Table 5 Effects of polyols (pH 7.0) on superoxide production by neutrophils

Table 6 Effects of acetoin and dihydroxyacetone on superoxide production by neutrophils at pH 7.0

Superoxide production (nmol per 107 cells per 30 min)a Untreated Polyols

Stimulus

Superoxide production (nmol per 107 cells per 30 min)a Untreated Acetoin Dihydroxyacetone

Control (HBSS) 1.6 µM PMA 1 µM fMLP

18.2 ± 3.0 81.9 ± 3.6 18.4 ± 1.6

Stimulus Control (HBSS only) 1.6 µM PMA 1 µM fMLP

13.9 ± 1.0 84.1 ± 11.4 19.2 ± 3.0

b

22.1 ± 2.0 66.3 ± 13.4b 37.9 ± 8.4b

Neutrophils were pre-incubated for 2 h in a mixture of polyols (25 mM glycerol, 8 mM mannitol, 7 mM erythritol, 6 mM D-glucitol), or buffer pH 7.0 before assay of superoxide production. a Values are the mean ± S.E.M. of four experiments. b Significant relative to untreated cells, paired two-tailed t-test, P < 0.05. Responses to PMA and fMLP have control values subtracted.

15.4 ± 1.7 90.0 ± 3.5 13.9 ± 1.4b

15.1 ± 2.2 89.4 ± 2.6b 11.4 ± 1.5b

Neutrophils were pre-incubated with 0.2 mM acetoin or dihydroxyacetone or buffer (untreated) for 2 h at pH 7.0 before assaying for superoxide production. a The results are means ± S.E.M. of four experiments. b Significant relative to untreated cells, P < 0.05, two-tailed paired t-test. The PMA and fMLP values have had controls subtracted.

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Table 7 The effects of pre-incubation of neutrophils with GABA at pH 5.5 on superoxide production Stimulus

Superoxide production (nmol per 107 cells per 30 min)a Untreated GABA

Control PMA fMLP

0.8 ± 0.6 2.9 ± 1.7 1.4 ± 0.6

1.1 ± 0.6 41.8 ± 12.5b 5.9 ± 2.8

a

Values are means ± S.E.M. of four experiments. Significantly different from the untreated cells, P < 0.05, paired twotailed t-test. Neutrophils were incubated for 2 h with 8 mM GABA or buffer (untreated) at pH 5.5 before superoxide production was assayed. Responses to PMA and fMLP have had control values subtracted. b

4. Discussion The results of this study do not support the hypothesis that early neutrophil influx into the lung is relatively low in infection by CNVG. We characterized the early inflammatory cell response to a strain of CNVG (TCS-SC1) [10], which was used previously in our rat models of pulmonary and cerebral cryptococcosis. These findings are similar to those reported after intra-tracheal inoculation of serotype D cryptococci into the same strain of rat as used in the present study [19], and also in a mouse model [3]. Time-course studies with high- and low-virulence strains of CNVN serotype A, suggested that rapid expansion of capsule size after inhalation and subsequent appearance of cryptococcal polysaccharide in the serum was inversely related to an early and intense neutrophil inflammatory response and was associated with increased virulence [20]. Neutrophil numbers also increased in the lung lavage fluid of mice in the first 12 h after intranasal inoculation with CNVN [21]. While we have shown that the neutrophil influx in response to CNVG is not compromised, we noted that small numbers of neutrophils were clustered around individual CNVG organisms, which remained extracellular despite an initially small capsule size, suggesting that neutrophil function was impaired. In the C. neoformans serotype D mouse model, clusters of neutrophils were described around degenerating macrophages associated with cryptococci [3], consistent with a direct cytotoxic effect of cryptococci. Candidate inhibitory or ‘toxic’ molecules produced by C. neoformans include enzymes, such as secreted phospholipase B, a proven virulence determinant [22] and proteases [23]. Dong and Murphy [7] reported that at pH 7, high-MW (> 30 000 Da) culture filtrates (which had not been heat inactivated) were not directly toxic to neutrophils. We reasoned that the toxicity we observed with vns and vgs is, therefore, due to non-enzymic components of < 30 000 Da, which we subsequently sought to characterize. Acetic acid and ethanol were the two most abundant metabolites in supernatants of both CNVN and CNVG; concentrations of acetate and ethanol in vns were approximately double those in vgs. We have previously reported similarly high levels of both metabolites in supernatants from a second serotype A clinical isolate of CNVN [9]. Although the effect

of exogenous ethanol on neutrophil function was not tested in this study, concentrations similar to those present in the cryptocococcal supernatants potentiate PMA-induced superoxide production via stimulation of membrane-associated protein kinase C [24]. In addition to acetic acid and ethanol, large quantities of polyphosphates were detected in both vns and vgs. In some yeasts, polyphosphate degradation occurs in response to environmental stresses [25], resulting in rapid glycogen mobilization for acid and ATP production. If cell growth were not proceeding, as in our packed cell suspensions, excess inorganic phosphate could be secreted to avoid formation of trehalose-6-phosphate, which is toxic to yeasts [26]. It is degraded to trehalose, a stress protectant. We have shown that the cryptococcal micro-environment in vivo is acidic, due to acetic acid production, and therefore, tested neutrophil functions after preincubation with cryptococcal supernatants adjusted to acidic pH (pH 5.5). This low pH was not cytotoxic per se, but was associated with complete inhibition of stimulated and unstimulated superoxide production. It is known that regulation of intracellular pH is essential for superoxide production, which can be impaired by exogenous acids, such as in abscesses, or by inhibition of Na+/H+ exchange [27]. In this respect, CNVG was less effective than CNVN (Table 1). In addition to diminished superoxide production, neutrophils preincubated with buffer or cryptococcal supernatants at low pH (5.5) were not capable of increased migration through bare filters in response to the chemoattractant, fMLP. Migration was also decreased in Transwells containing HBSS in the lower chamber, after pre-incubation with the vns or vgs. Previous work by Dong and Murphy [7] indicated that neutrophils in the presence of CneF from serotypes A and D migrated through bare filters to the same extent as controls to HBSS, but the response to fMLP was inhibited. Migration towards both HBSS and fMLP was inhibited in the presence of CneF from serotypes B and C [7]. In our experiments, though the presence of necrotic neutrophils in supernatantpreincubated cells may have interfered with the migration process, inhibition of the chemotactic response was clearly due to pre-incubation at low pH. However, under the same conditions, despite the presence of necrotic cells, adhesion to and/or neutrophil migration through monolayers of A549 epithelial cells was stimulated relative to untreated cells by preincubation in vns, and inhibited by preincubation in vgs. This result is similar to the findings of Dong and Murphy [7] using CneF as a chemoattractant, suggesting that components of cryptococcal supernatants may act directly on neutrophils as well as having chemoattractant properties (we did not test this latter function). The chemotactic and chemokinetic principle in CneF from CNVN appeared to be soluble GXM [7]. Capsular polysaccharides were present in both vns and vgs in concentrations of approximately 0.5 mg/ml (based on the levels of GXM antigen), but were not detected by MRS; at such concentrations, the broad signals associated with these high-molecular-weight compounds would be ex-

L. Wright et al. / Microbes and Infection 4 (2002) 1427–1438

pected to be lower than the detection threshold. Concentrations of 0.2–0.25 mg/ml of carbohydrate in CneF from CNVG were required to inhibit chemotaxis, and at least 0.4–0.5 mg/ml was required in CneF from CNVN for chemotactic effects [7]. Both of these effects were only demonstrable when the filtrate was present throughout the period of the chemotactic assay. Recently, mannoprotein (MP-4) purified from cryptococcal culture filtrates was shown to be directly chemotactic for neutrophils [28]. All of the neutrophil effects observed in our study were demonstrated in neutrophils exposed to supernatants which were both heatinactivated and washed off prior to assay of neutrophil function. GXM and/or MP-4, are therefore, unlikely to be responsible for increased migration through an epithelial monolayer of neutrophils preincubated with vns, and inhibition of migration after preincubation with vgs. In an attempt to further define the non-enzymic components of cryptococcal supernatants which affect neutrophil function, several of the most abundant compounds identified by MR spectroscopy were tested in comparison with vns and vgs for their effect on superoxide production at pH 5.5 and 7. At pH 7, a mixture of polyols, which were present in vns at concentrations higher than those in vgs, decreased the superoxide response to PMA and increased the response to fMLP, though this latter effect was not statistically significant. Neutrophils preincubated with vns exhibited an increased superoxide response to PMA and a similar response to fMLP compared with those preincubated in vgs. It is possible that the large amounts of ethanol in vns were sufficient to offset the inhibitory effect of polyols on PMA-stimulated neutrophil superoxide production (see above). The polyol, mannitol, is thought to protect cryptococci by scavenging reactive oxygen metabolites at sites of cryptococcal infection [29]. The MRS studies revealed two secreted metabolites present in vgs, acetoin and dihydroxyacetone, which were not observed in vns. The 13C chemical shifts of both compounds were in agreement with published values [30]. Dihydroxyacetone was detected only in the keto form, as evident from the 1H chemical shift [31] and absence of long-range 1 H–13C correlations to a hydrated carbonyl group. Acetoin and dihydroxyacetone are probably produced by diversion of carbon flux through the glycerol pathway rather than the ethanol pathway, as has been reported to occur under conditions of osmotic stress in Saccharomyces cerevisiae[32]. Acetoin can also act as a redox sink. Both compounds enhanced PMA-stimulated superoxide production and decreased that stimulated by fMLP, which is the opposite of the effect observed after preincubation of neutrophils with vgs and with the polyols. Under natural, as opposed to laboratory, conditions, these conflicting effects might not occur simultaneously, and may contribute to the adaptability of CNVG. For example, under stressful environmental conditions, the enzymes of the dihydroxyacetone pathway could be upregulated to metabolize the excess glycerol produced [32], whereas in the mammalian host, these enzymes would be suppressed, and polyol production would be more important

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in combating the oxidants produced by stimulated phagocytes such as neutrophils and macrophages. Our results confirm that several metabolites released by cryptococci into the extracellular environment affect neutrophil function. Both pro-inflammatory and anti-inflammatory activities are likely to be present at sites of cryptococcal infection. We propose that the cryptococcal products in the microenvironment of CNVN cause neutrophil necrosis and recruitment of host inflammatory and immune cells with subsequent containment of cryptococcal growth. On the other hand, inhibition of neutrophil function by products of CNVG at sites of infection results in survival of extracellular organisms, and local multiplication to form cryptococcomas. Our experiments illustrate that, as with Aspergillus[33], the effect of a putative virulence factor may be different when it is tested alone from when it is tested in combination with other molecules, which can alter its function to interfere with or stimulate different host responses.

Acknowledgements We would like to acknowledge the assistance of Sue Dowd with the in vivo measurements of pH. This work was supported by National Health and Medical Research Council grant number 990738.

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