Airway Epithelial Cell Culture and IDO Activity Assay
Mouse transformed airway epithelial cells (MTCCs) were obtained from Francisco DeMayo (Baylor College of Medicine, Houston, TX) and plated at a density of 3 × 104 cells per well in a 96-well plate with 200 μl media (RPMI 1640; ATCC, Manassas, VA). Adenovirus expressing either β-galactosidase (LacZ) or IDO, obtained from the University of Pittsburgh Vector Core, was added 24 hours after plating the cells. To measure kynurenine production, a high tryptophan-containing medium (RPMI 1640 containing 600 μM tryptophan) was added to the cells with the virus. Some wells were also treated with 1-methyl tryptophan (1-MT) (Sigma, St. Louis, MO). The 1-MT was dissolved in 1 M NaOH to create a stock concentration of 20 mM in media. Immediately before adding the cells, the 1-MT was further diluted to 1.5 mM. Cells were incubated at 37°C with virus for 48 hours, after which kynurenine was measured in the medium. We removed 160 μl of medium from each well and added 10 μl of 30% trichloracetic acid, and this was incubated at 50°C for 30 minutes to hydrolyze N-formyl–kynurenine to kynurenine. After centrifugation at 600 g for 10 minutes, the supernatant was transferred to a 96-well plate, and 100 μl of 1.2% (wt/vol) 4-(dimethylamine) benzaldehyde (Ehrlich reagent; Sigma) in glacial acetic acid were added. After 10 minutes at room temperature, the absorbance was read at 492 nm. A standard curve of kynurenine was generated to quantitate the concentration in virus-treated samples.
Whole Lung Extraction for IDO Activity
Whole lung was excised and frozen at −80°C until the assay was performed. Frozen lung was crushed using a liquid nitrogen–chilled mortar and pestle, and subsequently homogenized in 2 volumes of 0.14 M potassium chloride/0.02 M potassium phosphate buffer, pH 7.0. The homogenate was centrifuged at 30,000 × g for 30 minutes. The supernatant was removed and added to assay buffer (1:1), which contained 50 mM potassium phosphate buffer, 20 mM ascorbate, 200 μg/ml catalase, 10 μM methylene blue, and 400 μM L-tryptophan. The reaction was incubated at 37°C for 30 minutes, at which time the reaction was stopped and proteins were precipitated by the addition of 30% trichloracetic acid. The solution was then centrifuged at 1,000 × g for 5 minutes, and HPLC was performed to measure the production of kynurenine and the depletion of tryptophan.
HPLC was performed to measure kynurenine production, using samples prepared as already described. We injected 100 μl of sample into a C-18 column (250 × 4.6 mm, 5 μm; Waters, Milford, MA). The mobile phase consisted of 5 mM ZnCl2, 5 mM acetic acid, and 4% acetonitrile, pH 4.9, and samples were eluted at 1 ml/minute. The production of kynurenine was measured at an absorbance of 254 nm, whereas the depletion of tryptophan was measured in fluorescence (excitation, 360 nm; emission, 460 nm). Standards of kynurenine and tryptophan were run with the assay to establish the retention times and to allow for the determination of concentrations in the samples.
Semiquantitative and Quantitative RT-PCR
Total RNA was isolated using RNeasy Columns (Qiagen, Valencia, CA). The RNA was DNase-treated and reverse-transcribed into cDNA, using SuperscriptII (Invitrogen, Carlsbad, CA). Conventional RT-PCR was performed using SYBR PCR Master Mix (Bio-Rad, Hercules, CA). Real-time quantitative RT-PCR was performed using the iQ Supermix (Bio-Rad) and intron-spanning primers and probes (Applied Biosystems, Foster City, CA) for IDO, CC chemokine ligand 20 (CCL20), and mouse TLR9 (mTLR9), or SYBR PCR Master Mix and intron-spanning primers for human TLR9 (hTLR9) and β-actin. Forty cycles of PCR were performed using the Bio-Rad Chromo4 under universal cycling conditions: denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 1 minute. The level of gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels, and relative mRNA levels were determined according to the comparative cycle threshold method (ABI Prism 7700 Sequence Detection System, User Bulletin No. 2; Applied Biosystems). Briefly, the threshold cycle (CT) was determined for the gene of interest and GAPDH in each sample. The ΔCT was calculated for each sample by subtracting the CT of GAPDH from the CT of IDO. The ΔΔCT values were calculated by subtracting the ΔCT of Tg+ mice from the ΔCT of Tg− mice. The ΔΔCT values were transformed into absolute values, using the equation: 2T−ΔΔC. Primer sequences for assessing the expression of the human TLR9 (hTLR9) transgene were hTLR9 forward, 5′-CAGGGACAACCACCACTTCT-3′; and human growth hormone (hGH) reverse, 5′-GAGCAGGCCAAAAGCCAGGA-3′; and for β-actin, forward 5′-TCCTTCGTTGCCGGTCCACA-3′, and reverse 5′-CGTCTCCGGAGTCCATCACA-3′.
We purchased 6–8-week-old female BALB/cJ mice from Jackson Laboratories (Bar Harbor, ME). We purchased 6–8-week-old male B6.FVB-Tg(Itgax-DTR/EGFP)57LanJ mice from Jackson Laboratories and bred them with C57BL/6J mice. Transgene-positive pups were identified by PCR. All mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the University of Vermont. Mice were maintained on a 12-hour light/dark cycle and received food and water ad libitum. Our Institutional Animal Care and Use Committee granted approval for all studies.
Generation of Doxycycline-Inducible CC10-IDO Transgenic Mice
Transgenic mice were generated using three constructs: rCC10-tTS-hGHpA (suppressor; with rat CC10-tTS-human growth hormone for RNA stability), rCC10-M2-hGHpA (activator), and pTetOP-hGHpA. These constructs were a kind gift of Dr. Prabir Ray at the University of Pittsburgh. The hemagglutinin (HA)-tagged IDO sequence was created through PCR amplification of whole-lung cDNA generated from BALB/cJ mice. The primers used included: forward, 5′-CCGTCGACATGTACCCATACGACGTCCCAGACTACGCTGCACTCAGTAAAATATCTCCTAC-3′; and reverse, 5′-CGCGATATCCTAAGGCCAACTCATAAGAGCTTTCTC -3′. The forward primer contained a Sal
I site followed by the HA tag, and the reverse primer contained an Eco
RV site followed by a stop codon. The DNA sequence created through PCR was then cloned into the TA cloning vector (Invitrogen), the HA-IDO sequence was removed using Sal
I and Eco
RV, and the HA-IDO was subsequently placed in the multiple cloning site of the pTetOP-hGH vector, using the same enzymes. After all three constructs had been generated and the sequences verified, each was digested with Bss
HII to remove the promoter, transgene, and hGH polyA tail. The three separate transgenes were purified by three rounds of dialysis in injection buffer using 0.22-μm membranes (Millipore, Bedford, MA), and all three constructs were simultaneously microinjected into fertilized (C57BL/6 X C3H/HeN)F2
eggs. Transgenic mice were generated as previously described by the transgenic mouse facility at the University of Vermont (26
). Three lines of founders were obtained. However, only one of the three founders had inducible expression, and this founder was further backcrossed onto the BALB/cJ strain for at least eight generations. Initially, the founders were identified by slot blot, as previously described (27
), in which probes were generated by a restriction digest specific for tetracycline-controlled reverse transactivator (M2), tetracycline-controlled transcriptional silencer (tTS), and the tetracycline-regulated operator (TetOP). The probes for M2 were generated by digesting the CC10-M2-hGH plasmid with Hind
III and Bam
HI restriction enzymes, for tTS by digesting the CC10-tTS-hGH plasmid with Hind
III and Bam
HI, and for TetOP using Xho
I and Bam
HI. A 1% agarose gel was run, and the band of interest was excised and extracted from the gel, using a gel extraction kit from Qiagen. PCR was established subsequent to the slot blot procedure, and transgenic mice were identified through the PCR of genomic DNA for the incorporation of each transgene tTS, M2, and IDO. Primer sequences included: IDO forward, GAAGAGCCCTCAAATGTGGA; and hGH reverse, GAGCAGGCCAAAAGCCAGGA. For IDO, the forward primer was generated within the IDO sequence, and the reverse primer was specific for hGH, to differentiate between endogenous IDO and transgene IDO. Primer sequences for M2 included: forward, GACCAAAGTCATAAACGGCGC; M2 reverse, CGCGATGTGAGAGGAGAGCA; tTS forward, GAGCACAGCCACATCTTCAA; and tTS reverse, GAGTTGGCAGTTTCTCC.
Generation of TLR9−/− x CC10-hTLR9 Mice
CC10-hTLR9 mice were generated by excising the hTLR9 cDNA from the pCMV-SPORT6 plasmid (IMAGE clone identification 5,495,717), purchased from Invitrogen, with Eco
RV and Not
I. The ends of the hTLR9 fragment were filled in using a Klenow fragment. A plasmid containing the rat CC10 promoter, mutant IκBα, and hGHpolyA (27
) was cut with Sma
I and Bam
HI to liberate the IkBα fragment. The ends of the plasmid containing the rat CC10 promoter and hGHpolyA were filled in using a Klenow fragment, treated with calf intestinal alkaline phosphatase, and ligated with the hTLR9 fragment. The correct orientation of the hTLR9 insert was confirmed using Hind
III. The rCC10-hTLR9-hGHpolyA fragment was excised using Bss
HII, resolved on a 1% agarose gel, and purified using Elutip-D minicolumns, according to the manufacturer's instructions (Schleicher and Schuell, Keene, NH). The transgene was further purified by three rounds of dialysis against the injection buffer, using 0.22-μm membranes (Millipore), and microinjected into fertilized (C57BL/6 × C3H/HeN)F2 eggs. Transgenic mice were generated, and transgene integration was analyzed by slot blot, using a 502-base pair (bp) fragment from the human growth hormone sequence. Six transgenic founders were obtained, and three lines from these founders were backcrossed for six generations into TLR9−/−
mice on C57BL/6J. Transgenic mice were subsequently identified from genomic DNA obtained by ear-punch biopsy, using the following PCR primers: rCC10 forward, 5′-CACATTACAACATCAGCCCACATC-3′; and hTLR9 reverse, 5′-CTATTCGGCCGTGGGTCCCTGGC-3′.
Aspergillus fumigatus Crude Hyphal Extract Production
A. fumigatus lysates were generously provided by Dr. Kieren Marr (University of Washington School of Medicine, Seattle, WA), and were generated in a culture of Af293 isolate for 5 days at 37°C in RPMI-1640 plus 10% FCS. The hyphal mat was harvested, sequentially washed in PBS, and disrupted by vortexing with glass beads. To inactivate the extract, the slurry was subjected to 1% paraformaldehyde, and the protein concentration was measured using the Bradford Assay (Bio-Rad) according to the manufacturer's instructions. The product was concentrated using endotoxin-free dialysis membrane with a 10-kD pore size (Pierce Biotechnology, Rockford, IL) to achieve a final protein concentration of 800–1,000 μg/ml.
Aspergillus fumigatus Antigen Airway Exposure
Mice were anesthetized with isofluorane and administered 1 μg of A. fumigatus hyphal extract in 40 μl of PBS via oropharyngeal aspiration on days 0, 7, and 14. Mice were analyzed on day 19. For oropharyngeal aspiration, mice were anesthetized with inhaled isoflurane and suspended by their incisors on a 60° incline board with their tongues gently extended, and 25 μl of extract were delivered into the distal part of the oropaharynx and aspirated into the lower respiratory tract.
Assessment of Pulmonary Function to Measure Airway Hyperresponsiveness
Mice were anesthetized with 90 mg/kg of pentobarbital, tracheotomized, and mechanically ventilated for the assessment of pulmonary function using the forced oscillation technique, as previously described (28
). Briefly, the tracheotomy tube was connected to a computer-controlled, volume-cycled ventilator (FlexiVent; SCIREQ, Inc., Montreal, PQ, Canada), and mice were ventilated at 160–200 breaths/minute with a tidal volume of 0.2 ml and 3 cm H2
O positive end-expiratory pressure. Pressure, flow, and volume were measured to calculate the baseline and peak responses for airway resistance, tissue damping, and tissue stiffness (30
) after challenge with inhaled doses of saline or aerosolized methacholine (Sigma-Aldrich, St. Louis, MO) in saline, ranging from 3.125–50 mg/ml in half-log increments, as previously described (31
). The percentage of the response relative to baseline after each methacholine dose is reported.
Bronchoalveolar Lavage and Cell Enumeration
BAL fluid was collected after assessment of pulmonary function from euthanized mice by the instillation and recovery of 1 ml of 0.9% NaCl plus protease inhibitor cocktail (Sigma-Aldrich) into the lungs through the tracheal cannula, using a tuberculin syringe. BAL fluid was centrifuged at 400 × g, and the total cells in the pellet were resuspended in PBS and enumerated by counting with an Advia 120 Hematology System (Bayer HealthCare, Tarrytown, NY). For differential cell counts, 2 × 104 cells were centrifuged onto glass slides at 800 rpm. Cytospins were stained using the Hema3 kit (Biochemical Sciences, Inc., Swedesboro, NJ), and at least 500 cells were counted.
Histopathology and Morphometry
After euthanasia and bronchoalveolar lavage, the left lobes of lungs were instilled with 4% paraformaldehyde in PBS (4% PFA) for 10 minutes at a pressure of 25 cm H2O, and placed into 4% PFA at 4°C overnight for the fixation of tissue. Fixed lungs were then mounted in paraffin, and 7-μm sections were cut, affixed to glass microscope slides, and deparaffinized. Antigen unmasking was performed by boiling for 10 minutes in 10 mM sodium citrate buffer (pH 6.0). Slides were washed three times in distilled water and incubated in 3% hydrogen peroxide for 10 minutes, followed by two washes in water for 5 minutes each and then in PBS for 5 minutes each, and incubated in normal serum (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA) for 20 minutes. Slides were washed with PBS and stained with HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution in normal horse serum overnight at 4°C, and 100–400 μl of diluted antibody were added to each section. The remainder of the protocol was followed according to the manufacturer of the Vectastain ABC Kit. Multiple airways of similar size with a length/diameter ratio of less than 2:1 were assessed in each section by individuals blinded to the identity of sections. Sections were assessed for overall architecture, inflammation, and epithelial appearance.
Preparation, Stimulation, and ELISA Analysis of CD4+ Lymphocyte Suspensions
Single-cell suspensions were generated from spleens by passing the tissues through a 70-μm mesh, and lymphocytes were enriched in LSM Lymphocyte Separation Medium (MP Biomedicals, Irvine, CA). CD4+
T cells were isolated by positive selection, using CD4 magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Isolated CD4+
T cells were > 95% pure, as assessed by CD4 surface staining and FACS analysis. CD4+
T cells (4 × 106
cells/ml) were activated with 1 μg/ml of Aspergillus
hyphael extract in the presence of antigen-presenting cells (APCs) (4 × 106
cells/ml) from naive BALB/cJ mice, obtained through splenic T-cell depletion by negative selection, using antibodies to CD4, CD8, and Thy-1 and treatment with rabbit complement and mitomycin C, as previously described (32
). After 72 hours of stimulation in RPMI-1640 media (ATCC) containing 50 μM 2-mercaptoethanol and 10 μg/ml folic acid, supernatants were collected and analyzed by ELISA for IL-4, IL-5, IL-13, and IFN-γ, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Optical densities (ODs) from triplicate samples and duplicate standards were read using a PowerWavex
platereader (BioTek Instruments, Winooski, VT) at 450 nm, with background subtraction at 540 nm.
In Vitro Airway Epithelial Cell and CD4+ T-Cell Coculture
MTCCs were irradiated at 5,000 rad for 5 minutes, and 3 × 104 cells/well were plated in a 96-well plate. Twenty-four hours later, the medium was removed, and 1 × 109 viral particles (VP)/ml of adenovirus expressing IDO (AdIDO) or adenovirus expressing LacZ (AdLacZ) were added to each well for 24 hours. CD4+ T cells were generated by positive selection, as already described, and 1.5 × 105 cells/well were added to the MTCC culture after three washes. Anti-CD23 and anti-CD28 antibodies (B.D. Pharmingen, San Jose, CA) were added to a final concentration of 5 μg/ml and 1 μg/ml, respectively, to stimulate the CD4+ T cells polyclonally. Forty-eight hours later, cells were tritium-pulsed with 0.2 μCi/well 3H-thymidine (Perkin-Elmer, Boston, MA) and stimulated with 1 μg/ml each of anti-CD3 and anti-CD28. Twenty-four hours after the tritium pulse, the plates were harvested using a Tomtec Harvester 96 (Tomtec, Hamden, CT) according to the manufacturer's instructions, and liquid scintillation was used to measure CD4+ T-cell proliferation.
Whole-Lung Extraction and Flow Cytometry
The trachea of each mouse was cannulated, and the lungs were removed. Lungs were then inflated with 1 ml of RPMI containing 1 mg/ml of collagenase and 0.025 μl/ml of DNase. Lungs were further cut into pieces in 5 ml of RPMI containing collagenase and DNase, and incubated at 37°C for 30 minutes with gentle shaking. A catheter needle was used to break up the lung further, followed by a 30-minute incubation at 37°C with gentle shaking. The digested lung was then filtered through a 70-μM filter, and the supernatant was spun at 1,600 rpm for 5 minutes. The supernatant was removed, the pellet was resuspended in 1 ml of PBS followed by 12 ml of Gey's buffer, inverted to mix, and centrifuged at 4°C for 5 minutes at 1,600 rpm. The supernatant was removed and the pellet was resuspended in 8 ml of media, passed through a 40-μM filter, and centrifuged at 4°C for 5 minutes at 1,600 rpm. One wash was performed, the resulting pellet was resuspended in 5 ml of media, the cells were counted, and 1 × 106 cells were incubated in Fc block (anti-CD16/anti-CD32; B.D. Biosciences, Franklin Lakes, NJ) for 15 minutes on ice to prevent nonspecific antibody binding via Fc receptors. Cells were stained for 15 minutes on ice in 50 μl of antibody solution at the optimal concentration. Antibodies included CD4-PE-Texas Red (CalTag, Dorchester, UK), CD8α-AlexaFluor 647, TCRβ-FITC, and B220-PE (B.D. Biosciences). CD1-d tetramer, loaded with PBS57 and conjugated to phycoerythrin to stain natural killer T (NKT) lymphocytes, was obtained from the tetramer facility (Emory University Vaccine Center, Atlanta, GA) of the National institutes of Health. After 15 minutes, cells were washed, fixed, and subsequently distinguished using a LSR II FACS (Becton Dickinson, Franklin, NJ).
CpG DNA and Diphtheria Toxin Treatment
Mice were administered CpG DNA oligodeoxynucleotides (ODN 10104) or non-CpG (ODN 1827) (Coley Pharmaceutical, Ottawa, ON, Canada) via intraperitoneal injection or oropharyngeal aspiration. Diphtheria toxin (Sigma) was administered to CD11c-DTR mice at 50 μg/mouse, at a concentration of 1 mg/ml.
Western Blot for IDO and Actin
Lungs were frozen at −80° until analyses were performed. Powdered lungs were resuspended in Ripa buffer (25 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Sigma) and gently mixed for 1 hour at 4°C. The protein content was determined using the Bradford assay (Bio-Rad), according to manufacturer's instructions. Fifty miocrograms of protein were run on a 15% polyacrylamide gel, and proteins were then transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 hour in 5% nonfat dry milk. The primary antibody was incubated in 1% nonfat dry milk and 0.03% Tween-20 overnight at a dilution of 1:1,000. The IDO antibody was purchased from Chemicon (Billerica, MA), and the actin antibody was purchased from Santa Cruz Biotechnology. The secondary antibody was used at 1:1,000 in the same buffer as the primary antibody for 1 hour at room temperature.
Data were analyzed using the Student t test and two-way ANOVA, with Bonferroni correction for multiple comparisons. Statistical calculations were performed using GraphPad Prism 5 for Windows (GraphPad Software, Inc., La Jolla, CA). P < 0.05 was considered statistically significant.