Histone deacetylase activity is decreased in peripheral blood monocytes in patients with COPD

Study subjects were recruited at Renmin Hospital of Wuhan University in Wuhan, China. Criteria for recruiting patients with COPD included the following: 1) Diagnosis of COPD according to GOLD criteria with a ratio of post-bronchodilator forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ≤70% [13]; 2) age over or equal to 40 years; 3) cigarette smoke intake equal to or greater than 10 pack-years; and 4) chest CT scan indicating emphysematous abnormalities, such as barrel chest or central-lobular or pan-lobular emphysema. Exclusion criteria were as follows: 1) The presence of any other structural or functional lung disease, such as a past or current diagnosis of allergic rhinitis or atopy; 2) respiratory tract infection or exacerbation of COPD exacerbation no more than 6 weeks prior to screening; 3) asthma; 4) blood eosinophil count > 600 cell/mm³; 5) hospitalization for an acute COPD exacerbation no more than 3 months prior to screening; 6) clinically significant respiratory disease other than COPD; and 7) unstable cardiac condition. Inhaled salbutamol was permitted as needed so long as it was discontinued 24 hours prior to each study visit. Inhaled corticosteroids and oral sustained-release theophyllines were also permitted. Oral or parenteral corticosteroids at maximal doses equivalent to 10 mg/day of prednisone or 20 mg every other day were permitted. Patients who were already on oxygen therapy were included so long as their intake was < 15 hours per day and had been stable for at least 4 weeks prior to screening.

Recruitment criteria for control smokers were as follows: Voluntary cigarette intake of at least 10 pack-years with no history of lung disease or unstable cardiac conditions. The study also included healthy individuals with no history of smoking.

The studies were conducted in accordance with the Declaration of Helsinki, International Conference on Harmonisation Good Clinical Practice Guidelines. The study was approved by the Ethics Committee of the Wuhan University School of Medicine. All participants agreed to participate in this study and provided written informed consent before participating in the study.

We performed a prospective study with two visits on separate days. At visit 1, subjects provided written informed consent and were asked about their demographic information, respiratory symptoms by trained research nurse or doctor using standardized respiratory questionnaire. Physical examinations were performed at this time. FEV1 and FEV1 reversibility were tested. Smoking history information was also collected and quantified as pack-years. After visit 1, participants were asked to stop using all drugs and treatment. At visit 2, scheduled at least 2-4 days after visit 1, subjects underwent a spirometry post-bronchodilator test. Peripheral venous blood (20 ml) was extracted from each patient with a heparin syringe. The blood samples were isolated by density centrifugation within 4 hours, then PBMCs were stored at -70°C and serum were stored at -20°C, all samples were prepared less 3 month before analysis.

Spirometry tests were conducted according to ATS/ERS standardization guidelines for the performance of spirometry and assessment of lung volume using body plethysmography [15]. Typical practice in our laboratory incorporates quality control measures, including daily instrument calibration and review of study quality by a supervising technician and physician prior to interpretation. Flow-volume curves, FVC, relative FVC, FEV1, relative FEV1, and FEV1/FVC were measured using a spirometer (Vmax 229, Sensor-Medics, U.S.) and the best volume of the three manoeuvres was selected for data analysis. Data was expressed as the percentage of predicted normal values. Lung function test was conducted after inhaling 200 μg sabutamol (Glaxo Welcome, Chongqing, China).

Venous blood (20 ml) collected from each subject's ulnar vein was diluted 1:1 with sterile Hank's Balanced Salt Solution. PBMCs were isolated from the blood by density centrifugation as described previously [16]. Diluted venous blood (40 mL) was added on top of 20 ml of LymphoPrep (density, 1.077 g/ml) (Projen Biotechnik, Germany) and centrifuged for 20 minutes at 1,100 g at room temperature. The interface that contained the PBMC was collected and washed twice with phosphate-buffered saline (PBS). Cells were pelleted by centrifugation at 250 g for 8 min and stained with Kimura dye for determination of total cell number. Cell viability, as determined by trypan blue, was uniformly ≥95%.

Histones were extracted from PBMC nuclei using HCl and H₂SO₄ at 4°C following the method described by Ito [17]. Cells were microcentrifuged for 5 min and the cell pellets were extracted with ice-cold lysis buffer (10 mM Tris·HCl, pH 6.5/50 mM sodium bisulfite, 1% Triton X-100·10 mM MgCl₂/8.6% sucrose complete protease inhibitor mixture (Roche Molecular Biochemicals) for 20 min at 4°C. The pellet was washed repeatedly in buffer until the supernatant was cleared (centrifuged at 7,500 g × 5 min after each wash). The nuclear pellet was washed in nuclear wash buffer (10mMTris·HCl/13mMEDTA, pH 7.4) and resuspended in 50 μl of 0.2 M HCl and 0.2 M H₂SO₄. The supernatant was mixed with 1 ml of ice-cold acetone and left overnight at -20°C. The sample was microcentrifuged for 10 min, washed with acetone, dried, and diluted in distilled water. The protein concentrations of the histone-containing supernatant were determined using a Bradford protein assay kit (Bio-Rad).

HDAC activity in PBMC nuclear extracts was assessed using an HDAC Activity/Inhibitor Screening Assay Kit (Cayman, U.S.). An acetylated lysine substrate is incubated with samples with HDAC activity. Deacetylation sensitizes the substrate such that treatment with the HDAC developer in the second step releases a fluorescent product. The assay was performed exactly as recommended by the manufacturer. Fluorescence signal was detected at 460 nm using a fluorometric plate reader.

Serum levels of interleukine-8 (CXCL8) were measured by sandwich ELISA (Jingmei Biotech Co., Ltd., Shenzhen, China) according to the manufacturer's instructions.

Demographic data from all participants are presented in Table 1. There were 23 healthy volunteers, among them 10 were habitual cigarette smokers and 13 were never smokers. All 26 patients with COPD were habitual cigarette smokers, of whom 7 were in GOLD stage 1, 8 in GOLD stage 2, 8 in stage 3, and 3 in stage 4 (Table 1,2). No significant differences were observed between COPD cases and controls in terms of age, height, and body weight.

HDAC activity in the PBMCs of COPD patients was significantly decreased by 40% as compared to that in healthy non-smokers (13.06 ± 5.95 vs. 21.39 ± 4.92 μM/μg, p < 0.001). HDAC activity in PBMCs of healthy smokers was also 40% lower than that observed in healthy non-smokers (12.50 ± 4.27 vs. 21.39 ± 4.92 μM/μg, p < 0.001). (Figure 1)

HDAC activity in PBMCs was decreased as the smoking levels (pack-years) increased, (Figure 2). In COPD patients who were heavy smokers (≥ 40 pack-years), HDAC activity in the PBMCs was 40% lower than that in COPD patients who smoked fewer than 40 pack-years(Figure 3).

Higher serum CXCL8 levels were found in stable COPD patients than in non-smoker controls. In addition, increased serum CXCL8 levels were negatively correlated to HDAC activity in PBMCs (r = -0.468, p < 0.01) (Figure 4). Patients who smoked more than 40 pack-years had higher serum CXCL8 levels than those who had smoke less than 40 pack-years.

No correlation was found between the severity of airway obstruction and HDAC activity or serum CXCL8 level (all p > 0.05).(Table 3).