Human study subjects
Study participants were randomly selected from outpatients and inpatients of Xinhua Hospital, Shanghai Jiao Tong University. Twenty participants with allergic asthma (10 women, 10 men) and twenty participants (10 women, 10 men) were healthy controls. Asthma was diagnosed from common symptoms and pulmonary functions . The severity of asthma was evaluated on the basis of the Global Initiative for Asthma criteria , both mild and moderate asthmatics were enrolled. The skin-prick test (SPT) was performed using a standard panel of 11 allergens, a 15 mm diameter skin wheal response was defined as positive. In the four weeks prior to the study, asthmatic participants were permitted to be treated with inhaled glucocorticoid but not systemic steroids.
The healthy control subjects with age and sex matched to allergic asthma participants were reported no allergic diseases, were negative SPT and had not received oral or intravenous steroids in the previous 4 weeks before the study.
All subjects were fully informed about the purpose and nature of the studies, which were approved by the medical ethics committee of Xinhua Hospital. Written informed consent was obtained from the patients for publication of this report and any accompanying images.
Peripheral blood T lymphocytes
Isolation and purification
Twenty milliliters of fresh human peripheral blood was obtained from allergic asthmatics and healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (d = 1.077 mg/ml; Nycomed Pharma AS, Roskide, Denmark). The purified blood T cells were subsequently obtained using T Cell Negative Isolation Kit (Invitrogen Dynal AS, Oslo, Norway) according to the manufacturer’s instruction. The purity of T cells was 90-95%.
The purified peripheral blood T cells were nucleofected with pCMV-myc-LAT, pCMV-myc or LAT-siRNA plasmids (nucleofect with three kinds of plasmids in healthy controls and pCMV-myc-LAT, pCMV-myc plasmids in asthma patients) using the Human T Cell Nucleofector® Kit (Amaxa, Lonza, Germany). The protocol was done according to the manufacturer’s instruction.
The nucleofected T cells were activated with anti-CD3 (1 μg/ml, BD Bioscience) and anti-CD28 (1 μg/ml, BD Bioscience) and cultured for 24 h at 37°C with 5% CO2. Cells were harvested for Western Blot analysis and the supernatants were collected and preserved at −20°C for ELISA.
Male, 6-8-week-old Wistar rats from Sinobritish Sipprbk Lab Animal Ltd (Shanghai, China) were maintained under pathogen-free conditions at the animal center of Xinhua Hospital, Shanghai and were age- and sex-matched within each experiment. Mice studies were approved by the Xinhua Hospital Animal Care and Use Committee.
Sensitization and airway challenge
Wistar rats were sensitized twice, with an interval of 7 days, by the intraperitoneal (i.p) injection of 0.5 ml of 2 mg chicken ovalbumin (OVA) (Grade V, Sigma-Aldrich, shanghai, china) bound to 200 mg aluminum hydroxide Al(OH)3 (Sigma) in saline. Simultaneously, 6 × 109 heat-killed Bordetella pertussis bacilli were administered intraperitoneally as an adjuvant. From day 15 to day 28, rats were exposed to aerosolized OVA (1% in saline) or saline alone (Control groups) for 20 minutes once a day. The aerosol was generated with a nebulizer (PARIBOY N037, PARI, Starnberg, Germany) and was drawn into the exposure chamber containing the awake animals. Rats were euthanized (50 mg/kg pentobarbital, i.p.) 24 h after the final challenge, serum, bronchoalveolar lavage fluid (BALF) and lungs were sampled.
Lung T cells isolation and activation with or without TSA
Lung lymphocytes were acquired by Lymphocytes Separation Medium (HISTOPAQUE 1083, Sigma, St. Louis, MO, USA). T lymphocytes were purified from mononuclear cells by nylon wool filtration. The purity of T cells was > 85%, as assessed by flow cytometry with anti-CD3-FITC antibody (eBioscience, San Diego, CA, USA). The purified lung T cells were incubated with or without TSA (2.5 ng/ml) for 24 h. After incubation, the supernatants were collected, pooled per stimulation for cytokine production analysis, and cells were harvested for either total protein and RNA isolation or ChIP assay.
The level of rats IL-4 and IFN-γ in BALFs and serum of immunized and non-immunized rats, and in the supernatants of cultured lung T cells with or without TSA were measured and quantified by ELISA following the manufacturer’s instructions (ELISA kit, invitrogen). As well as the production of IL-4 and IFN-γ in the human peripheral blood nucleofected T cells from allergic asthmatics and healthy controls.
Quantitative real-time PCR
Total RNA in human peripheral blood T cells and rat lung T cells was extracted using TRIzol reagent (invitrogen) and 1 μg of RNA was reversed transcripted using cDNA synthesis kit (TaKaRa, Dalian, China). The primer specific for LAT (forward, 5’-GAGGATGTGGATGGAGAGGA-3’; reverse, 5’-CTGTAGGCA AGGCAGAGGTC-3’, which produced a 143 bp product) and GAPDH (forward, 5’-GCAAGTTCAACGGCACAG-3’; reverse, 5’-GCCAGTAGACTCCACGACAT-3’, which produced a 140 bp product), were designed and synthesized by Beyotime Institute of Biotechnology, Shanghai, China.
The quantitative real-time PCR was performed on an ABI PRISM 7500 Fast Real-Time PCR System (ABI, USA), and SYBR Green (TaKaRa, Dalian, China) was used as a double-stranded DNA-specific fluorescent dye. GAPDH was used as a housekeeping gene for standardizing LAT mRNA expression. After normalization of the data according to the expression of β-actin mRNA, relative levels of LAT and GAPDH were calculated using the 2—△△Ct method .
Lung T cell and human nucleofected T cell protein extracts were prepared. Approximately 20–30 μg of protein was subjected to 10% SDS/PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). After transfer, the membranes were blocked by 5% fat-free milk in TBST (20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20) for 1 h at room temperature (RT) and incubated with primary antibodies against LAT (1:750) (Cell Signal), HDAC1 (1:750) (Cell Signal, Danvers, MA, USA), acetyl-H3 (1:20000) (Millipore Corporation), acety-H4 (1:2000) (Millipore Corporation) or dimethyl-H3K9 (1:500) (Millipore Corporation) overnight at 4°C in the TBST, and then incubated with an HRP-labeled secondary antibody (1:1000) (Beyotime Institute of Biotechnology, Shanghai, china) for 2 h at RT. Incubation with anti-GAPDH (1:1500) (Abcam, San Francisco, CA 94105, USA) served as a loading control. The blots were visualized by chemiluminescence with ECL Western blotting detection reagents (Millipore Corporation).
The ChIP assay was carried out using a ChIP assay kit (Millipore) according to the manufacturer’s protocol with minor modification. Cultured lung T cells (~8 × 106) were cross-linked by 0.4% formaldehyde at 37°C for 5 min, and then excess formaldehyde was quenched by addition of glycine at a final concentration of 0.125 M. Sonication was performed on ice using a Bioruptor sonicator to shear the cross-linked DNA to an average length of 200–1000 bp. Supernatants of the samples were collected and diluted 10 fold in ChIP dilution buffer (20 μl of each was reserved as total input control) followed with preimmunoprecipitation clearing with 80 μl of a Protein A Agarose/Salmon Sperm DNA −50% Slurry for 30 minutes at 4°C with agitation. Immunoprecipitation was performed with 4 μl of anti-acetylhistone H3, anti-acetylhistone H4 anti-acetylhistone H3 or anti-dimethylhistone H3 (K9) and incubated overnight at 4°C with rotation. Immune complexes were collected with 60 μl protein A agarose/salmon sperm DNA for 60 min at 4°C with rotation and washed twice with low salt buffer, once with high salt buffer, once with LiCl buffer, and twice with TE Buffer. The immune complexes were eluted twice from the antibody by with 250 ul elution buffer. The elutes and the input were heated at 65°C for 6 h to reverse histone-DNA croosslinks by the addition of 20 μl of 5 M NaCl. DNA was extracted using a DNA Mini Preparation Kit (Beyotime Institute of Biotechnology, Shanghai, china). 2 μl of each of the purified DNA was used as template for 32 cycles of PCR amplification. The PCR products were analyzed on 1.5% agarose gel. The following primers of rat LAT gene, covering the 5’-flanking region (−75/+23), were used: 5’-TGAGGAGCCTGATGATTTCC-3’; 5’-GCTG TACCTGCCTTTCTTGC-3’. Non-specific IgG (Beyotime Institute of Biotechnology, Shanghai, China) was served as the negative control in the assay.
All data were expressed as mean ± SD. Differences between groups were calculated for statistical significance using the Student’s T-test. A P-value ≤ 0.05 was considered as statistically significant.