Peptide synthesis
STAT-6-inhibitory peptides (STAT6-IP or IP) and STAT6-control peptides (STAT6-CP or CP) were synthesized by the University of Calgary Integrated Peptide Services (Calgary Alberta, Canada). Peptides are comprised of a derivative of the TAT protein transduction domain, YARAAARQARA [20]. This sequence is coupled to an 8 amino acid sequence surrounding tyrosine 641 in murine STAT6. In the STAT6-IP the tyrosine residue is phosphorylated (GRG*YVSTT) and in the control STAT6-CP peptide, this residue is replaced by a phenylalanine (GRGFVSTT). Peptides were amidated at the carboxyl terminus and purified by RP-HPLC and analyzed by MS. Peptide sequences, uptake, stability in vivo and inhibitory effects of the STAT6-IP have been detailed previously in an acute OVA-induced [17] and chronic ragweed induced models of AAD [21].
Animals
Four to six weeks old BALB/c mice were obtained from Harlan-Spraque Dawley (Indianapolis, IN). Mice were bred and housed in a conventional animal facility at the Meakins-Christie Laboratories. For each experimental condition a minimum of 6–8 animals was used. All studies followed the Canadian Council of Animal Care (CCAC) guidelines and were approved by the Animal Care Committee of McGill University, animal protocol number 4658.
Peptide Vaccination
Beginning on the third day of life, 50 µg of either STAT6-IP or STAT6-CP, or PBS, was administered intranasally (IN) in a volume of 10 µL daily for 5 days [19].
Allergen sensitization and challenge
For chronic ragweed-induced AAD: Awake 4–6 week old animals were sensitized 5 days per week for 5 weeks by local instillation of 10 µl in each nare of 1% (10 mg/ml) ragweed (Giant Ragweed, extracted from Ambrosia trifida, Sigma-Aldrich, St Louis MO) in PBS as previously described [18]. After a two week rest, mice were challenged with 1% (10 mg/ml, dry weight) Amba I (Short Ragweed, extracted from ambrosia artemisiifolia pollen grains, Greer, Lenoir NC) in PBS once per day for 5 consecutive days and assayed 24 hours following the final challenge.
Assessment of airway hyperresponsiveness (AHR) by methacholine challenge
Total respiratory resistance was measured 24 hours after ragweed challenge using a small animal ventilator (FlexiVent, SCIREQ, Montreal, Qc Canada) as described in detail elsewhere [17, 21]. Briefly, mice were deeply anesthetized with xylazine hydrochloride (NovaPharm, Toronto, On, Canada) followed by sodium pentobarbital (Sandoz, Montreal Qc Canada, CEVA, Santé Animale, France) and paralyzed with pancuronium bromide. Heart rate was monitored by EKG to ensure adequate anesthesia throughout the procedure. Mice were ventilated quasi-sinusoidally with a tidal volume of 8 ml/kg and a respiratory rate of 150 breaths/min. Methacholine was given by nebulizer (Aeroneb, SCIREQ, Montreal, Qc) at doses of 3.125, 25, and 50 mg/ml and maximal resistance were obtained for each graded dose of methacholine.
Histological analysis and differential cell count in bronchoalveolar lavage fluid (BAL)
Pulmonary histopathology was performed as described previously [22]. Briefly, lungs were slowly inflated with 1 ml formalin, isolated, and then placed entirely in formalin. The specimens were embedded in paraffin, and 0.5 µm sections were cut. Representative images are shown at 100 × magnification. Immediately post-mortem, lungs were lavage twice in situ with 0.75 ml of ice-cold saline. The returned fractions were pooled and centrifuged. The pellets were used for total and differential cell counts as previously described [22]. Briefly, red blood cells were lysed using ACK lysing buffer (PharM Lyse™, BD Biosciences, Mississauga, ON). Cells were spun onto glass slides and stained using the Diffquick method (Dade Behring Inc IL). Differential cell counts were obtained manually under light microscopy. 40⋅ power fields were counted per slide and means obtained.
Measurement of ragweed-specific IgE in serum
Blood was obtained from mice post-challenge by post-mortem intracardiac puncture. Ragweed-specific IgE was assayed as described previously [17, 21, 23]. Specific IgE was quantified by ELISA (Pharmingen OptEIA™ IgE Kit, San Diego CA) with the following modifications: serum was incubated overnight at 4ºC with protein G sepharose beads (Amersham Bioscience, Piscataway, NJ) before loading onto ELISA plates that had been previously coated with short ragweed. Standards were plated as per OptEIA protocol. Samples were incubated overnight at 4ºC and ELISA performed as per protocol. The specific antibodies were quantified with mouse IgE ELISA sets (BD Biosciences Pharmingen, San Diego, CA).
Splenocyte culture and cytokine assessment
Single-cell suspensions were prepared from the whole spleen as described previously [19, 21]. Briefly, erythrocytes were lysed by ACK lysing buffer and the washed splenocytes were resuspended at 5 × 106 cells/ml in complete RPMI-1640 medium with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate. Splenocytes were then cultured for 4 days at 37 °C with 5% CO2 in the presence of Amba I (10 mg/ml). Supernatants were stored at -80ºC before quantifying cytokine levels by ELISA. Murine IL-4, IL-10, IL-13, TGFβ, and IFNγ levels in supernatants of the ragweed-stimulated cultured splenocytes were quantified using ELISA kits purchased from eBioscience Inc (San Diego, CA) as per manufacturer’s instructions.
T cell suppression assay
CD4+ T cells were purified from spleens of ragweed sensitized mice and incubated with carboxyfluorescein succinimidyl ester (CFSE; 5 µM) for 8 minutes at room temperature [19]. Cells were then washed and resuspended in media for 15 minutes. CFSE-labeled CD4+ T cells (responder T cells) were mixed with CD4+ T cells, isolated as described above, from STAT6-IP treated or sham control mice in ratio of 1:1 to 1:8 and cocultured in 24-well plates. Cells were stimulated with 10 mg/mL ragweed in the presence of 2 × 105 purified CD11c+ DCs from allergen naïve mice. After 3 days, cells were harvested, and CFSE dilution was analyzed by means of flow cytometry [19].
Adoptive transfer of CD4+ T cells or CD11c+ DCs
For adoptive transfer experiments, 5 ⋅ 106 MACS purified CD4+ T cells, from IP- or CP-vaccinated or PBS sham vaccinated BALB/c mice, were injected into the tail vein of naïve BALB/c recipients in a volume of 50 µL of normal saline [19]. Twenty-four hours later recipient mice were sensitized and challenged with ragweed as per protocol. In other experiments 5 ⋅ 105 MACS purified CD11c+ DCs from these same donor mice were injected intratracheally into naïve BALB/c recipients in a volume of 50 µL of normal saline [19]. Twenty-four hours later these mice were sensitized and challenged with ragweed as per protocol [19].
In vivo treatment with the anti-TGFβ antibody
Animals were treated with 50 µg anti-TGFβ pan-neutralizing antibody or isotype control IN every second day during sensitization with the allergen as previously described [19]. Monoclonal pan-anti-TGFβ1,-β2,-β3 (MAB 1835) and normal mouse IgG1 isotype control (MAB002) were purchased from R&D System (Minneapolis, MN).
RNA extraction and Real-Time RT-PCR
To assess mRNA levels of inflammatory genes in lung tissues, the total cellular RNA was extracted from the lungs using RNeasy Mini Kit, according to the manufacturer’s protocol (Qiagen Inc, Toronto, On, Canada), and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Real-time quantitative PCR was performed with a LightCycler System (Roche Applied Sciences, Indianapolis IN) using QuantiTect SYBR Green (Qiagen Inc, Toronto, On). Each target was quantified using four 10-fold serial dilutions of standards prepared from PCR amplicons that had been gel purified and quantified using a Fluorochem 8000 imaging system and AlphaEase software (Alpha Innotech, San Jose, CA). Values were then normalized to GAPDH that had been reverse transcribed, PCR amplified, and quantified. The following mouse primers were used [19]: Muc-2, sense 5'-GCT GAC GAG TGG TTG GTG AAT G-3' and antisense 5'-GAT GAG GTG GCA GAC AGG AGA C-3', Muc-5ac, sense 5'-CAG CCG AGA GGA GGG TTT GAT CT-3' and antisense 5'-AGT CTC TCT CCG CTC CTC TCA AT-3'. Eotaxin-1 (CCL11), sense 5'-GGG CAG TAA CTT CCA TCT GTC TCC-3' and antisense 5'-CAC TTC TTC TTG GGG TCA GC-3'. GAPDH, sense 5'-GCC ATG GAC TGT GGT CAT GA-3' and antisense 5'-TTC ACC ACC ATG GAG AAG GC-3'. IDO, sense 5'-GTA CAT CAC CAT GGC GTA TG-3' and antisense 5'-CGA GGA AGA AGC CCT TGT C-3'.
Flow cytometric analysis
Surface markers and FoxP3 were evaluated by flow cytometry [19]. Splenocytes cultured for 4 days with Amba I were stimulated for 5 h with murine anti-CD3 (0.5 µg/ml; clone 2C11, BD Pharmingen, Mississauga, On, Canada) and monensin (GolgiStop; BD Biosciences, Mississauga, On, Canada), according to the manufacturer’s instructions. Cells were then washed, permeabilized with saponin (Perm/Wash; BD Biosciences), and fixed in formaldehyde and PBS (Cytofix/Cytoperm; BD Biosciences) for 30 min. Cell surface markers were stained with labeled rat anti-mouse CD4, CD25, TGFβ, or Foxp3 on ice for 30 min and then washed with PBS and analyzed on a FACSCalibur (BD, San Jose, CA). Ten thousand cells were counted from each sample and data were analyzed with BD flow cytometer and Cell Quest Software. Cells were also stained with respective isotype control antibodies.
Statistical analysis
The results represented ± SEM of six to eight replicates per experiment and each experiment was repeated at least three times. The data was analyzed using one-way analysis of variance followed by Tukey’s post hoc tests for individual group comparisons. A value of p < 0.05 was considered significant.