Immunosuppressive substances in Aspergillus fumigatus culture filtrate

J Infect Chemother (2003) 9:114–121 DOI 10.1007/s10156-002-0227-1

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2003

ORIGINAL ARTICLE Akira Watanabe · Katsuhiko Kamei · Toshikazu Sekine Mayumi Waku · Kazuko Nishimura · Makoto Miyaji Takayuki Kuriyama

Immunosuppressive substances in Aspergillus fumigatus culture filtrate

Received: July 23, 2002 / Accepted: December 10, 2002

Abstract Invasive aspergillosis has become a serious problem in clinical practice, but the actual factor that confers virulence on the fungus has not been thoroughly elucidated. To identify and isolate the immunosuppressive substances produced by the fungus, the bioactivity of culture filtrates was assessed, and analyses of the culture filtrates were carried out. Culture filtrates from different strains of Aspergillus fumigatus were assessed for their effect on human polymorphonuclear leukocytes and murine macrophages. To assess their activities in vivo, their effect on the survival of mice infected by the fungus was also studied. Subsequently, the composition of the culture filtrates was analyzed by gas chromatography-mass spectrometry. The analyses revealed that the culture filtrates contained gliotoxin at concentrations of 3 to 4 µg/ml, and some other unidentified compounds. The bioactivities of the culture filtrates were similar to those of gliotoxin. The fungal culture filtrate reduced the survival of infected mice, but the filtrate itself did not cause the death of mice. However, all the bioactivities could not be accounted for by gliotoxin itself. These results indicate that gliotoxin in the culture filtrates may be responsible for part of the immunosuppressive activity, but some other components produced by A. fumigatus contribute, in an additive or synergistic manner, to the virulence of the fungus. Key words Asperqillus fumigatus · Gliotoxin · Macrophages · Polymorphonuclear leukocytes · Virulence factor

Introduction Aspergillus fumigatus is the most frequent cause of invasive aspergillosis in immunocompromised patients.1 The occurrence of such infections has dramatically increased among immunocompromised patients.2 This disease can also develop in immunocompetent hosts.3–5 Various toxic factors produced by A. fumigatus have been assumed to be virulent in the fungal infection;6–11 however, the factors that actually play an important role in the pathogenesis of aspergillosis have not been identified as yet.1,12 Among these substances, gliotoxin, a secondary metabolite produced by the pathogen, has been shown to exert potent bioactivities on various mammalian cells.13–17 To date, its role in the virulence of the fungus has remained questionable, because it becomes evident in the fungal culture filtrate only after long culture.17,18 In a previous report, we demonstrated that A. fumigatus culture filtrate caused drastic morphological changes in murine macrophages.19 We surmised that gliotoxin accounted for the cytotoxicity of the culture filtrate, but information has been insufficient to support this hypothesis. To identify and isolate immunosuppressive substances present in the culture filtrate, we investigated the in-vitro and in-vivo bioactivities of the culture filtrate and analyzed its composition by gas chromatography.

Materials and methods A. Watanabe (*) · T. Kuriyama Department of Respirology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba 260-8670, Japan Tel. ⫹81-43-222-7171; Fax ⫹81-43-226-2176 e-mail: [email protected] K. Kamei · K. Nishimura · M. Miyaji Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan T. Sekine · M. Waku Department of Sciences for Medicinal Resources, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Fungi Clinical isolates of A. fumigatus (IFM 47439, from a patient with invasive pulmonary aspergillosis; IFM 47450 and IFM 49824, from patients with allergic bronchopulomonary aspergillosis) were used in this study. All isolates were stored and have been maintained at the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University. Each isolate was cultured on potato dextrose agar (Difco Laboratories, Detroit, MI, USA) slants at 35°C for 7

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days for sporulation. The spores were collected with distilled water (plus 0.05% Tween 20) and washed two times. The spore suspension was adjusted to 5 ⫻ 104/ml in RPMI 1640 (Sigma Chemical, St. Louis, MO, USA). Preparation of fungal culture filtrate Thirty milliliters of the spore suspension was poured into 150-mm Petri dishes (Nalge Nunc International, Roskilde, Denmark) and cultured at 37°C in a humidified 5% CO2 incubator. After 24 h, the cultures were collected, filtersterilized through a 0.22-µm filter (Millipore, Bedford, MA, USA), and stored at ⫺85°C until used. The pH of the culture filtrates was determined using a pH meter (Yanagimoto, Kyoto, Japan). Preparation of human polymorphonuclear leukocytes To separate polymorphonuclear leukocytes (PMNs) from blood cell populations, we employed Mono-Poly Resolving Medium (Dainihon Pharmaceutical, Osaka, Japan) according to the manufacturer’s directions. Briefly, Mono-Poly Resolving Medium and fresh, heparinized blood from healthy volunteers was settled in centrifugal tubes. The tubes were centrifuged and PMNs were collected. The viability of PMNs, as measured by the Eosin Y dye exclusion test, was more than 95%. Effect of the culture filtrates on polymorphonuclear leukocytes Migration Chemotaxis of PMNs was assessed using modified Boyden chamber20 devices (Chemotaxicell; Kurabo, Osaka, Japan). Briefly, PMNs were preincubated with RPMI 1640 containing culture filtrate at different concentrations for 1 h at 37°C and washed with RPMI 1640. The viability of PMNs was measured as described above. N-Formyl-L-methionyl-Lleucyl-L-phenylalanine (Sigma) was adjusted to 10⫺9 M and was dispensed in 24-well microplates (500 µl/well), as the chemoattractant. A Chemotaxicell (3-µm pore size) was placed on each of the wells. The PMNs (5 ⫻ 104) in 200 µl RPMI 1640 were placed in the device and incubated at 37°C and 5% CO2 in a humidified atmosphere for 90 min. The filter of the device was fixed and treated with May-Giemsa stain for PMNs. Superoxide anion production The release of superoxide anion from PMNs was assessed based on the reduction of cytochrome c (Sigma), by a technique modified from that of Nakagawara and Minakami.21 Briefly, PMNs were exposed to culture filtrate as described above, and washed with Hank’s balanced salt solution (HBSS), and the viabilities were determined. Then PMNs (5 ⫻ 105) were suspended in 1 ml of HBSS containing

cytochrome c (0.1 µmol), with or without phorbol myristate acetate (PMA; 0.01 nmol; Sigma), and incubated in a shaking water bath at 37°C for 1 h. Then, the supernatants were read at 550 nm with the reference wavelength at 540 nm in a spectrophotometer. Preparation of macrophages Murine peritoneal macrophages were collected according to the procedure modified from that of Levitz and DiBenedetto.22 Eight-week-old male BALB/c mice (Charles River Japan, Yokohama, Japan) were anesthesized with ether, and their peritoneal cavity was lavaged with RPMI 1640. The resident peritoneal cells obtained were rinsed and adjusted to 2 ⫻ 105/ml in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA). The suspension was dispensed in flat-bottomed 96-well microplates (100 µl/well) (Nalge Nunc International). The viability of nucleated cells was more than 87%. Preparations of the cell suspensions stained with Diff-Quik (International Reagents, Kobe, Japan) showed more than 70% macrophages, and the peritoneal cells were used as resident peritoneal macrophages. Effect of the culture filtrates on macrophages Macrophages were challenged with the culture filtrates at different concentrations and cytotoxicity was examined. Cell viability was tested by WST-8 (2-[2-methoxy-4nitrophenyl]-3-[2,4-disulfophenyl]-2H-tetrazolium monosodium salt) reduction assay23,24 (TetraColor ONE; Seikagaku, Tokyo, Japan). The culture filtrates, at different dilutions, were added to the wells in which the macrophages had settled. After 18–24 h of incubation, 10 µl of TetraColor ONE was added to each well and the plates were incubated for an appropriate period of time. Absorption at 450 nm was determined, setting the reference wavelength at 630 nm, using a microplate reader. Mouse model of aspergillosis Culture filtrate of A. fumigatus (IFM 47450) was prepared afresh as described above. Six-week-old male ddY mice (Gokita Breeding Service, Tokyo, Japan) were divided into three groups: (1) a group of mice was intraperitoneally injected with 2.5 ⫻ 108 A. fumigatus spores (IFM 47450) in 1 ml of the culture filtrate, (2) another group of mice was injected with the same number of spores in 1 ml of RPMI 1640, and (3) the control group of mice was injected with 1 ml of the culture filtrate. Each group consisted of ten mice. Upon death or humane killing, their internal organs were removed for pathological examination. Fractionation of the culture filtrates by molecular weight The culture filtrates were centrifuged at 6800 g at 4°C for 100 min in a centrifugal filter device (Centricon YM-3; Millipore). The filtrate obtained was used as the fraction

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that contained low-molecular-weight substances (less than 3 kDa) (fraction L). The retentate was reconstituted with RPMI 1640 to the original volume of the culture filtrate, and centrifuged at 6800 g at 4°C for 100 min through the same device. This procedure was repeated twice, and the retentate finally obtained was used as the fraction containing high-molecularweight substances (more than 3 kDa) (fraction H). Fraction H was reconstituted to the original volume with RPMI 1640. Both fractions and the original culture filtrate were filtersterilized through the 0.22-µm Millipore filters, and were added to the macrophages to determine their cytotoxicity, as above. Extraction by chloroform Each culture filtrate (400 to 500 ml) was added to an equal volume of chloroform and shaken vigorously. Then, the aqueous and chloroform layers were separated. The extraction was repeated three times. The chloroform layer was dried over Na2SO4 overnight at room temperature. After filtration, chloroform was evaporated under reduced pressure until dryness at a temperature below 45°C to obtain the chloroform extract. Then, the extract was dissolved in dimethylsulfoxide (DMSO) (Wako Pure Chemical, Osaka, Japan) and reconstituted with RPMI 1640, so that the final volume of the extract was the same as that of each culture filtrate. The concentration of DMSO in this sample was 1.5%. The effects of this sample and the aqueous fraction on macrophages were assessed as described above. DMSO diluted in RPMI 1640 at 1.5% was also examined. Gas chromatography-mass spectrometry Gas chromatography-mass spectrometry (GC-MS) analyses of the samples were carried out with 5890 Series II, 5971A (Hewlett-Packard, Palo Alto, CA, USA). The extract from the chloroform fraction was dissolved in a methanol/chloroform mixture (1 : 1) at a concentration of 1 mg/ml. Two microliters of this sample solution was injected into the GC system provided with a 30 m ⫻ 0.252-mm inner diameter, 0.25-µm film thickness DB-1 column (J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas, at a flow rate of 35 ml/min. The temperatures of the detection port and injection port were 280°C and 250°C, respectively. The MS used a 3-min solvent delay and a scan rate of 1.6 scan/s. The chromatograms were recorded and analyzed, and areas under peaks of interest were integrated for quantitative analysis. Identification of the peaks in the chromatograms was carried out using a library search program (Hewlett Packard G1030A Version A MS Chemstation software). Gliotoxin Authentic gliotoxin (Sigma) was dissolved in 100% ethanol to a concentration of 1 mg/ml, adjusted to the appropriate

concentrations with RPMI 1640, and tested on PMNs and macrophages as described above. The gliotoxin solution was also given intraperitoneally to the infected mice, and the effect on their mortality was investigated. Regarding GC-MS analysis, gliotoxin was dissolved in a methanol/ chloroform mixture and injected into the system to elicit its spectrum. Solutions containing known concentrations of gliotoxin were also injected to correlate the quantity of gliotoxin with the area under its peak. Statistical analysis Statistical significance was determined with two-factor factorial analysis of variance and Bonferroni’s multiple comparisons test. To assess differences regarding mortality among the groups of mice, we employed the Cox-Mantel test. P values less than 0.05 were considered significant. Ethics All mice were cared for in accordance with the rules and regulations set out by the Prime Minister’s Office of Japan. Animal protocols were approved by the Special Committee on Animal Welfare of our institution.

Results Chemotaxis of PMNs The numbers of migrating PMNs decreased in a concentration-dependent manner when the cells were exposed to the culture filtrates (Table 1). Chemotaxis was nominally, but not significantly, retarded by exposure to 0.05% culture filtrate. Migration of PMNs exposed to 0.25% to 0.5% culture filtrate was significantly suppressed (P ⬍ 0.001). Oxidative burst by human PMNs The amount of superoxide anion released from PMNs decreased in a concentration-dependent manner when the cells were exposed to the culture filtrates (Table 2). The oxidative burst by PMNs stimulated with PMA was significantly suppressed by pretreatment with 3.125% to 6.25% culture filtrate (P ⬍ 0.001). Without stimulation, the sponTable 1. Effect of the culture filtrates on polymorphonuclear leukocyte (PMN) migration Culture filtrates

Control

0.05%

0.25%

0.5%

IFM 47439 IFM 47450

11.0 ⫾ 1.4 12.3 ⫾ 2.1

9.25 ⫾ 1.0 8.5 ⫾ 3.8

3.0 ⫾ 0.8* 7.5 ⫾ 2.1

0 ⫾ 0* 0.3 ⫾ 0.5*

* P ⬍ 0.001 vs control Values are means ⫾ SD of the number of PMNs that migrated through the pore of the filter. Four randomly chosen microscope fields were examined. The other culture filtrate (IFM 49824) exhibited similar activity

117 Table 2. Inhibition of oxidative burst of PMNs by the culture filtrates Culture filtrates Control IFM 47450 IFM 49824

3.125%

6.25%

Table 4. Cytotoxicity of the culture filtrate and its L and H fractions

12.5%

105.1 ⫾ 20.6 89.6 ⫾ 4.3 56.9 ⫾ 5.4* 21.5 ⫾ 3.9* 71.6 ⫾ 7.7 43.8 ⫾ 4.1* 20.2 ⫾ 0.9* 7.1 ⫾ 0.3*

* P ⬍ 0.001 vs control Values shown are means ⫾ SD of the amount (nmol) of superoxide anion produced by 1 ⫻ 106 PMNs. The experiments were done in triplicate. Each amount was calculated as the difference in absorption from that of the sample without phorbol myristate acetate (PMA). A similar tendency was observed when the other culture filtrate was used (IFM 47439)

Original culture filtrate Control 0.5% 1% 5%

0.44 0.20 0.17 0.20

⫾ ⫾ ⫾ ⫾

0.02 0.03*;** 0.01* 0.02*

Fraction L (MW ⬍3 kDa) 0.44 0.43 0.19 0.18

⫾ 0.02 ⫾ 0.04*** ⫾ 0.02* ⫾ 0.01*

Fraction H (MW ⬎3 kDa) 0.44 0.52 0.46 0.43

⫾ ⫾ ⫾ ⫾

0.02 0.07 0.02 0.01

* P ⬍ 0.001 vs control. ** P ⬍ 0.001 vs 0.5% fraction L*** Values are means ⫾ SD optical density of experiments carried out in quadruplicate. This table shows the results of one representative strain (IFM 47450). MW: molecular weight

Table 3. Effect of the culture filtrates on the viability of macrophages Culture filtrates Control IFM 47439 IFM 49824

0.1%

0.2%

0.5%

0.43 ⫾ 0.01 0.36 ⫾ 0.08 0.18 ⫾ 0.02* 0.17 ⫾ 0.01* 0.43 ⫾ 0.01 0.43 ⫾ 0.04 0.36 ⫾ 0.06 0.17 ⫾ 0.01*

* P ⬍ 0.001 vs control Values are means ⫾ SD of the optical density of experiments done in triplicate. The optical density was used as an index of viability after a linear colorimetric response, showing a good correlation with the number of viable macrophages, was confirmed (data not shown). The other culture filtrate (IFM 47450) showed similar activity. The mean ⫾ SD of the optical density of the sample without the macrophages was 0.183 ⫾ 0.026

taneous release of superoxide anion was negligible (data not shown). The exposure of PMNs to the filtrates at these concentrations (up to 12.5%) for 1 h did not lower the viability of the PMNs. Viability of murine peritoneal macrophages At concentrations of 0.2% to 0.5%, the culture filtrates were significantly cytotoxic for macrophages (P ⬍ 0.001; Table 3). At these concentrations, almost all the macrophages were assumed to have been killed, because the optical density was similar to that of the sample without macrophages. Mortality of infected mice The mortality of the infected mice was significantly accelerated by the culture filtrate (P ⬍ 0.05; Fig. 1). The culture filtrate itself did not influence the mortality of the mice. Pathological and histological examination of all dead mice showed dissemination of A. fumigatus. The fungal hyphae grew in the brain, lungs, liver, kidneys, and intestine.

Fig. 1. Mortality was compared among three groups of mice. Each group consisted of ten mice. Each mouse in group 1 (closed circles) was intraperitoneally injected with Aspergillus fumigatus spores (IFM 47450) in culture filtrate. All mice in group 2 (closed squares) were injected with the spores in RPMI 1640, and mice in group 3 (open circles) were given only the culture filtrate. The culture filtrate significantly reduced the survival of the infected mice, but the filtrate itself did not cause the death of the mice. *P ⬍ 0.05 vs group 3 (open circles) on day 28

Molecular weight and solubility in chloroform Examination of the fractionated culture filtrates showed that fraction L retained cytotoxicity on macrophages, whereas fraction H had nominal activity (Table 4). The original culture filtrate showed higher cytotoxicity than its fraction L at the concentration of 0.5% (P ⬍ 0.001). After extraction with chloroform, the cytotoxicity of the residual, aqueous fraction was significantly curtailed (P ⬍ 0.001; Table 5). DMSO (0.15%) exhibited marginal cytotoxicity. This finding indicates that the chloroform extract itself showed cytotoxic activity.

pH of the culture filtrates Gas chromatography-mass spectrometry The pH of all culture filtrates was found to be approximately 7.4 (data not shown); therefore, the results described above were confirmed not to be due to the acidification of the culture medium.

Many peaks were detected when the chloroform fraction was analyzed by GC-MS (Fig. 2). These peaks were investigated with the MS system in electron-impact ionization mode, and

118 Fig. 2. A Gas chromatogram of the chloroform extract from the culture filtrate of IFM 47439 strain is shown. Numerous peaks were detected, including those for desthiogliotoxin (retention time, 13.9 min), phthalic ester (retention times, 11.9, 12.3, and 15.5 min), and unidentified substances (retention times, 14.2, 14.4, 14.9 min, and others). Phthalic ester was assumed to correspond to impurity in the organic solvent. B The mass spectrum of the peak detected at the retention time of 13.9 min is shown

Table 5. Cytotoxicity of the chloroform extract of the culture filtrate Original culture filtrate Control 1% 5% 10%

0.58 0.15 0.15 0.16

⫾ 0.11 ⫾ 0.01* ⫾ 0.00* ⫾ 0.00*

Chloroform extract

Aqueous fraction

DMSO (1.5%)

0.58 ⫾ 0.15 ⫾ 0.14 ⫾ 0.15 ⫾

0.58 ⫾ 0.60 ⫾ 0.62 ⫾ 0.60 ⫾

0.58 0.53 0.54 0.49

0.11 0.00* 0.00* 0.00*

0.11 0.01 0.02 0.02

⫾ 0.11 ⫾ 0.01 ⫾ 0.03 ⫾ 0.00

* P ⬍ 0.001 vs control Values are the means ⫾ SD of the optical density of experiments done in triplicate. The results of a representative strain (IFM 47450) are shown. The final concentrations of dimethylsulfoxide (DMSO) were 0 to 0.15%

the library search system. A peak representing desthiogliotoxin was noted when the fractions of each culture filtrate were analyzed. Chromatograms of the fractions derived from IFM 47439, IFM 47450, and IFM 49824 revealed the peak of desthiogliotoxin at retention times of 13.9, 13.7, and 14.0 min, respectively. The GC-MS showed that the culture filtrates also contained phthalic ester and some other compounds, which were not found in the database.

Comparison between gliotoxin and the culture filtrates Gliotoxin and the culture filtrates showed similar bioactivities on PMNs and macrophages. Gliotoxin significantly retarded chemotaxis at the concentration of 0.01 µg/ml (P ⬍ 0.001). The production of superoxide anion was significantly suppressed by gliotoxin at the concentration of 0.25 µg/ml (P ⬍ 0.001). The viability of macrophages was significantly curtailed by 0.02 µg/ml gliotoxin (P ⬍ 0.001; Table 6). Regarding the experiment with infected mice, gliotoxin (5 µg/mouse) did not alter their mortality (data not shown). GC-MS of gliotoxin revealed that the peak representing desthiogliotoxin was at the retention time of 13.9 min, and that it was the same molecule as that disclosed in the analysis of the culture filtrates (Fig. 3). This result verified that gliotoxin changed to desthiogliotoxin under the conditions employed for GC-MS analysis. The concentration of gliotoxin in each culture filtrate was calculated from the area under the peak. The filtrates prepared from strains IFM 47439, IFM 47450, and IFM

119 Fig. 3. A Gas chromatogram of authentic gliotoxin at 1 mg/ml in the organic solvent. B The mass spectrum of the peak detected at the retention time of 13.9 min coincided with that of desthiogliotoxin

Table 6. Effect of gliotoxin on the viability of macrophages Agents

Control

Gliotoxin (µg/ml)/ethanol (%) 0.01/1 ⫻ 10⫺5

Gliotoxin Ethanol

0.52 ⫾ 0.02 0.53 ⫾ 0.00 0.52 ⫾ 0.02 0.50 ⫾ 0.04

0.02/2 ⫻ 10⫺5

0.03/3 ⫻ 10⫺5

0.40 ⫾ 0.01* 0.53 ⫾ 0.05

0.23 ⫾ 0.01* 0.58 ⫾ 0.08

* P ⬍ 0.001 vs control Values are means ⫾ SD of the optical density of experiments done in quadruplicate. Gliotoxin was dissolved in 100% ethanol at 1 mg/ml; therefore, the concentration of ethanol in medium containing 0.01 µg/ ml gliotoxin was 1 ⫻ 10⫺5%. The mean ⫾ SD of the optical density of the sample without the macrophages was 0.220 ⫾ 0.006

49824 contained gliotoxin at concentrations of 3.92, 3.22, and 3.45 µg/ml, respectively.

Discussion Our study disclosed five major findings. First, the culture filtrates prepared by the method used in the present study

showed immunosuppressive activities; namely, they suppressed PMN migration and the oxidative burst induced by PMNs, and killed macrophages. Second, the in-vitro activities of gliotoxin were similar to those of the culture filtrates. Third, each culture filtrate made from IFM 47439, IFM 47450, and IFM 49824 strains contained approximately 3 to 4 µg/ml gliotoxin. Fourth, the culture filtrates also contained other substances, which have not been identified to date. Fifth, the culture filtrate reduced the survival of mice infected with A. fumigatus. Gliotoxin is known as a secondary metabolite produced by A. fumigatus and other fungi;25 it has many immunosuppressive activities. Gliotoxin can induce apoptotic cell death in various mammalian cells,13,26 and it suppresses the migration of PMNs, as well as superoxide anion release from PMNs.14–16,27 In the present study, the culture filtrates exhibited in-vitro bioactivities similar to those of gliotoxin. These results are congruent with those of previous reports. Moreover, the quantities of gliotoxin proposed to be required to attain immunosuppressive effects on PMNs were compatible with our results. Murayama et al.27 demonstrated that gliotoxin significantly restricted the migration

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of and superoxide anion production by PMNs at concentrations of 0.03 and 0.3 µg/ml, respectively. In the present study, PMN migration was suppressed by 0.25% to 0.5% culture filtrate, which contained 0.01 to 0.016 µg/ml gliotoxin, while the oxidative burst was inhibited by 3.125% to 6.25% culture filtrate, which contained 0.11 to 0.25 µg/ml gliotoxin. Taken together, the toxic effects observed in the present study would be, at least partly, attributable to gliotoxin. However, the results of our study also lead us to conclude that components other than gliotoxin found in A. fumigatus culture filtrates play an additive role in virulence. A. fumigatus culture filtrate itself reduced the survival of the infected mice. A. fumigatus may produce components which participate in the development of the actual infection. Gliotoxin could be one of them. We made a fresh culture filtrate for this animal experiment, and did not analyze it because of its small volume. Therefore, whether it contained gliotoxin was not confirmed. Sutton et al.28 proposed that gliotoxin produced by A. fumigatus hyphae during infection exacerbated the pathogenesis of aspergillosis. In their study, gliotoxin was administered once to each mouse by intraperitoneal infusion at a dose of 100 µg, and this treatment rendered resistant mice susceptible to infection by A. fumigatus. However, in our study, the dose of gliotoxin in the culture filtrate given to each mouse was assumed to be approximately 3 to 4 µg. This amount of gliotoxin is assumed to be too small to cause the death of infected mice. This finding supports the view that gliotoxin may not be the only component responsible for the various immunosuppressive activities observed in our study. Other results of the present study also support this hypothesis. Almost all the macrophages were killed when exposed to 0.2% to 0.5% culture filtrate (i.e., a concentration of gliotoxin less than 0.02 µg/ml), whereas authentic gliotoxin did not exhibit a similar activity at 0.02 µg/ml. In addition, the cytotoxicity of the culture filtrates was weakened by the removal of high-molecular-weight substances. These findings also provide evidence that the culture filtrates contain some other compounds, which are distinct from gliotoxin, but whose activity is additive or synergistic. In other studies, putative virulence factors were also assumed to exist,6–10 including undefined substance(s) which inhibited neutrophil and alveolar macrophage functions.29–31 These studies claimed that some of the factors might restrict host defenses at the local level, thereby contributing in some degree to the colonization and infection caused by A. fumigatus. However, whether these factors contributed to the bioactivities noted in the present study has not been confirmed as yet. In the present study, some substances for which there was no information in the database of the GC-MS were detected. In addition, the GC was originally designed to detect low-molecular-weight substances that are volatile and hydrophobic, such as gliotoxin. It cannot be ruled out, therefore, that in the culture filtrates some compounds, as yet still undetected, exhibited synergistic activity but were not toxic themselves. Additional studies are in progress to identify these components and to clarify their activities.

In conclusion, the present study demonstrates the in-vivo and in-vitro bioactivities of A. fumigatus culture filtrate, and sheds light on the pathogenesis of infection by A. fumigatus. Acknowledgments This study was done in the project “Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)” through the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government in 2002. A part of this study was also supported by SFIF fund, NPO Biomedical Science Association.

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