HNEC primary cultures
The HNEC culture conditions were adapted from a culture model originally developed with human tracheobronchial cells [36,37]. Nasal polyps were sampled in patients with nasal polyposis during ethmoidectomy. HNEC cultures were prepared as previously described [11]. In brief, each polyp was placed overnight at 4°C in PBS-antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 μg/ml amphotericin B and 100 mg/ml gentamicin) solution containing 0.1% pronase (Sigma-Aldrich, L'Isle d'Abbeau Chesnes, France). After rinsing, the cells were dissociated in a 0.25% trypsin solution (Invitrogen, Cergy Pontoise, France) and 106 cells were plated in 12-mm insert wells (Corning BV, Schiphol-Rijk, The Netherlands) with microporous membranes coated with type IV collagen (Sigma-Aldrich) and incubated at 37°C in 5% CO2. After 24 h, the culture medium was removed from the apical compartment to obtain an air-liquid interface. The basal compartment was filled with 1 ml of the HNEC culture medium, consisting of DMEM/F12 with 2% Ultroser G (Sigma-Aldrich) with antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml gentamicin). The HNEC culture medium was changed daily and the electrophysiological properties of the HNEC were checked twice weekly using a microvoltmeter with concentric electrodes (EVOM® World Precision Instruments, Stevenage, UK).
Fungal strain culture conditions and mycelium mycotoxin extraction
We used Aspergillus fumigatus IP 2279-94, originally isolated from a patient with invasive aspergillosis. The strain was cultured on Potato Dextrose Agar (PDA) medium (Sigma-Aldrich) at 37°C for 7 days in the dark. Conidia were collected by gently scraping with spatula and rinsing the PDA Petri dish surface with tryptone salt solution (tryptone 0.1%, 0.8% sodium chloride, 0.1% Tween 80). The suspension was filtered through sterile compresses to remove hyphae. Two hundred and fifty μl of conidial suspensions (2.105 conidia/ml) were used to inoculate three 500 ml Erlenmeyer flasks each containing 100 ml minimal Eagle's medium (Sigma-Aldrich) supplemented with 5% sterile fetal calf serum, and flasks were then placed in a shaking incubator at 180 rpm for 3 days at 25°C and 37°C, as previously described [38]. After filtration of collected medium through Whatman N°1, the pH of the filtrate was checked and the fungal cultures were extracted with 70 ml chloroform for 4 days at room temperature. The organic phase was evaporated under vacuum at 50°C using a rotary evaporator. The residue was taken up in 200 μl methanol; this suspension was then filtered through a disposable filter with 0.45 μm pores before injection (20 μl) into the chromatograph apparatus.
Chemicals
Verruculogen, fumagillin, and helvolic acid from Sigma-Aldrich were used without further purification (purities higher than 90%). The sources of the other secondary metabolites used as authentic standards were: P.M. Scott, Health Canada, Ottawa, Canada (fumigaclavine A), J.W. Dorner, National Peanut Research Laboratory, ARS-USDA, Dawson, USA (fumigaclavine C), C. Avendano. Universidad Complutense, Madrid, Spain (fumiquinazoline F), P.G. Mantle, Imperial College, London, UK (TR2) and H. Osada, RIKEN Institute, Wako, Japan (Tryprostatin A). Fumitremorgin C was purchased from Alexis Biochemicals.
Fractionation of culture filtrates by HPLC and identification of secondary metabolites
The residual extract was fractionated and fungal metabolites were analyzed by HPLC-DAD using a 150 mm × 4 mm Zorbax ODS 5 μm C18 column (Bischoff, Leonberg, Germany) in an HPLC chromatograph apparatus (Kontron, Milan, Italy). HPLC analysis was performed based on a modified Frisvad chromatographic method [39], with a flow rate of 2 ml/min and a linear elution gradient of acetonitrile (solvent B) in 0.2% acetic acid (solvent A). Analysis started with 10% of solvent B, which was increased to 50% after 30 min, and then to 90% four min later, maintained at 90% for a further 4 min, then decreased over 7 min to 10%, and finally maintained at 10% over 1 min. Compounds were identified by comparison with mycotoxin standards analyzed in the same manner. The first fractionation was performed as follows: four 10-minute fractions (F1-4) and one 6-minute fraction (F5) were collected after DAD detection. Fractions were evaporated and taken up in 25 μl DMSO. Ten μl of the DMSO-solubilized HPLC fractions were diluted in 90 μl of HNEC medium, 15 μl of this solution were diluted in 1.5 ml of HNEC medium, and 750 μl of this final solution were used to immerse one HNEC well. After an initial biological activity screening, the positive fraction was split in the same way into two-minute fractions (F'1-5). Fractions were evaporated and taken up as described above.
Biological activity of the fractions on HNEC
All experiments were performed on 14-day HNEC cultures when cell differentiation was established and stable. All tests were performed in duplicate and each experiment was repeated at least three times with different HNEC cultures. Interactions between HNEC and fungi were evaluated using bioelectrical properties of HNEC cultures, i.e. transepithelial resistances (Rt) and potential differences (Vt), which measure intercellular junction permeability and trans-cellular ionic transport, respectively [40]. For 14-day cultures, the usual range of Rt and Vt is 600 to 800 ohms/cm2 and -20 to -40 mV, respectively. The day before the assay (referred to as T0), the HNEC inserts were immersed in 750 μl of HNEC culture medium. After 30 min to balance electrodes, the Rt and Vt were recorded. After the first measurement, the culture medium was removed from the apical side to recover the air-liquid interface. Twenty-four hours later, 750 μl of the solution containing the tested product were added to the apical side of HNEC cultures and the electrophysiological values were measured at 30 minutes and 3 hours. For a given well, the results were expressed as a percentage variation from the T0 measurements. Calculations were performed using the mean +/- SEM obtained from at least three different cultures.
Ability of different A. fumigatus isolates to produce verruculogen
Sixty-seven A. fumigatus strains of the Pharmacology Toxicology Laboratory New Collection (NCPT, INRA Toulouse, France) were examined for their ability to produce verruculogen. These strains were selected from various human, animal, plant and environmental sources and geographical regions. Verruculogen production by A. fumigatus strains was measured in autoclaved grains of Ardente, a variety of durum wheat. One hundred grams of wheat grains were moistened with 100 ml of sterile distilled water for 4 days at 4°C, before sterilization. Twenty grams of grain were placed in a 140 mm diameter Petri dish. Each dish was inoculated with 250 μl containing 2 × 105 conidia and incubated at 25°C for 13 days. The fungal cultures were extracted with 100 ml chloroform as described above.
Mycotoxin extraction from conidia
For detection of mycotoxins associated with conidia, and after having demonstrated that production of verruculogen was effective with every A. fumigatus isolate, we pursued experimentation with the NCPT strain 13, which has been used for numerous experiments with mycotoxin production (also available under number NRRL 35693 at the Northern Region Research Laboratory, USDA-ARS, Peoria, Ill, USA). Large amounts of A. fumigatus conidia of the NCPT strain 13 were produced on Czapek Yeast Extract Agar (CYA) medium for 14 days at 37°C. Conidia were collected by inverting CYA Petri dishes over a collecting flask. Conidia (10 mg) were gently mixed for 1 min in 2 ml of 0.9% sodium chloride solution containing 0.1% Tween 80. After harvesting, the suspension was filtered through a porosity 2 filter-funnel to remove hyphae and extracted for 4 days by shaking in 10 ml of chloroform at room temperature. The extract was evaporated under vacuum at 50°C on a rotary evaporator. The crude extract was taken up in 500 μl of methanol, and this solution was filtered through a 0.45 μm Teflon filter before liquid chromatography and mass spectrometry analysis.
HPLC-MS and MS-MS analysis
The LC-MS-MS instrument used was an LCQ DUO Ion Trap from Thermo-Finnigan. The LC separation was done on a 150 × 2.1 mm C18 Kromasil column (Interchim, Montluçon, France) using a 30-minute linear gradient of 10% to 90% acetonitrile in the presence or absence of 0.1% formic acid (presence for positive ionization mode). Electrospray ionization (ESI) was performed at 4.5 kV and the capillary temperature was set to 250°C using a gas flow of 70 ml/min and an auxiliary gas flow of 20 ml/min. MS and MS-MS tuning were performed on 5 μM authentic verruculogen and the MS-MS collision was set at 48%. Whole mycelium and conidial extracts were analyzed in negative and positive ESI mode in full scan and MS-MS mode with an m/z range of 80 to 700. The mass spectra of the positive fraction and verruculogen standard were obtained by infusion in positive ESI mode.
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