Association of autophagy related gene polymorphisms with neutrophilic airway inflammation in adult asthma

Study subjects recruitment

We enrolled 408 asthmatic patients and 201 healthy normal controls (NCs) from Ajou University Hospital (Suwon, Korea) to a case-controlled study. Asthma was diagnosed at the first evaluation based on a history of respiratory symptoms as well as the results of airway reversibility and hyperresponsiveness to methacholine following the Global Initiative for Asthma guideline. Severe asthma was defined as asthma that requires treatment with high dose inhaled corticosteroids plus a second controller and/or systemic corticosteroids to control it or that remains “uncontrolled” despite this therapy, follows American Thoracic Society guidelines [15]. All asthma patients were recruited when the disease was stable on their regular medications without any viral or bacterial airway infection. NCs were healthy individuals who had no history of asthma symptoms. All the subjects were provided written informed consents prior to participating in this study.

Atopy was defined as one or more positive reactions on a skin prick test with 55 common inhalant allergens (Bencard Co., Brentford, UK) with histamine and saline controls. Methacholine bronchial challenge tests were performed as previously described using doubled doses of methacholine (0.075 to 25 mg/mL) [16]. The methacholine PC20 value (the concentration of methacholine needed to produce a 20% decrease in forced expiratory volume in 1 second [FEV1]) was determined by interpolation from a dose-response curve.

Sputum induction and blood collection

Sputum induction was performed as previously described [17]. Briefly, asthma subjects were pretreated with 200 μg salbutamol through a metered dose inhaler. The subjects then were inhaled nebulized sterile 3% saline solution for 20 minutes through an ultrasonic nebulizer (Omron Co., Kyoto, Japan). Expectorated sputum was collected into a petri dish after excluding the saliva. Simultaneously with sputum collection, venous blood was collected into acid citric dextrose containing tubes (BD Falcon, Franklin Lakes, NJ, USA) for genomic DNA preparation and Vacuette tubes (Greiner Bio-One, Monroe, NC, USA) for serum collection.

Assessment of sputum neutrophil count

Each sputum sample in petri dish was weighted and transferred into a 50 mL polystyrene tube. Four times volume (v/w) of freshly prepared dithiothreitol 0.1% (DTT, Sigma, St. Louis, MO, USA) diluted in distilled water was added to each sputum tube followed by incubation at 37℃ for 20 minutes with occasionally gentle vortex to dissociate the disulfide bonds of the mucus. The reaction was stopped by added phosphate buffer saline (PBS) in a volume equal to the sputum plus DTT solution. The tubes were centrifuged at 1,500 rpm for 5 minutes. The cell pellet was resuspended in 50 mL PBS and filtered through a 40 μm nylon filter (Millipore, Bedford, MS, USA). Total cell count and cell viability were determined by staining with Trypan Blue (Sigma) and observing under an inverted microscope. To assess differential cell count, a cytospin slide of each sample was prepared, fixed with methanol and performed hematoxylin and eosin stain. All slides were overread (total 500 cells) and the samples with more than 80% squamous cells were excluded from analysis. Sputum neutrophil count was expressed as percentage of neutrophils among non-squamous cells in each sample.

SNP selection and genotyping

Genomic DNA was prepared from peripheral blood samples using Puregene Blood Core Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. We screened for SNPs located on promoter and 5’-untraslated region (UTR) of ATG5 and ATG7 from 40 healthy Korean volunteers by direct sequencing. Three SNPs of ATG5 including –769T>C, –335G>A in promoter region and 8830C>T in 5’UTR, were selected. Two SNPs of ATG7, including –100 A>G in promoter and 25108G>C in 5’UTR, were selected. ATG5 and ATG7 are located on chromosome 6 and 3, respectively. The information of ATG5 and ATG7 polymorphisms are shown in Table 1. Minor allele frequency of all selected SNPs was over than 5%. Target SNPs were genotypes from all genomic DNA samples of study subjects using a primer extension method and the SNaPshot ddNTP primer extension kit (Applied Biosystems, Foster City, CA, USA) with primers shown in Table 2.

Table 1.

The single nucleotide polymorphisms of ATG5 and ATG7 in this study

Table 2.

The amplifying and extension primers and probes for ATG5 and ATG7 polymorphisms genotyping

Preparation of ATG5 constructs for promoter assays

A 3201 base-pair DNA fragment that contains ATG5 –769T>C and –335G>A was amplified using the following primers: forward primer 5’-CTCTATCGATAGGTACCAAGTGATGGTTAGGGTTCATGG-3’ and reverse primer 5’-GATCGCAGATCTCGAGGCCCTCCGTGTTCTGCCTAA-3’. The 3’ end of each primer (underlined) contains homologous sequences with ATG5 and the 5’ ends contain 15-16 bases homologous to each end of the linear pGL3-basic vector (Promega, Madison, WI, USA). Linear vector was prepared using Kpn1 and Xho1 restriction enzymes (Takara, Shuzo, Japan). The PCR amplified fragments were gel-purified using Expin Gel kit (GeneAll, Seoul, Korea) and then cloned into the linear pGL3-basic vectors using In-fusion HD cloning kit (Clontech Laboratories Inc., Mountain View, CA, USA) following the manufacturer’s manual.

Cell culture

Human airway epithelial cell (A549) and human mast cell (HMC-1) were purchased from American Type Culture Collection (Manassas, VA, USA). A549 cells were cultured in RPMI-1640 and HMC-1 cells were cultured in Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G sodium and 100 μg/mL of streptomycin sulfate (Gibco). The cells were maintained at 37oC in 5% CO₂ humidified atmosphere.

Endogenous mRNA expressions of ATG5 and ATG7

Total mRNA was extracted from various cell lines using Easy-Blue reagent (Intron Biotechnology, Seongnam, Korea) according to the manufacturer’s protocol. cDNA was synthesized by reverse transcription (RT)-PCR from 3 μg of total mRNA using MMLV-RTase (Promega) under optimized reaction condition (reaction buffer: 50 mM Tris-HCl pH 8.3, 17 mM KCl, 3 mM MgCl₂, 10 mM DTT, 2 mM dNTP, 0.5 U/mL RNase inhibitors [Promega] at 42˚C for 1 hour 10 minutes and extanded at 70˚C for 10 minutes). The mRNA expression levels of ATG5 and ATG7 was evaluated by PCR using the specific primers (ATG5: forward 5’-TCTCTTGACTGCAGTACCTTTTC-3’ and reverse 5’-CGTGTCTGATACTGTAAGCCTTT-3’; ATG7: forward 5’-CGACGCTCAGTAGCCTGTAG-3’ and reverse 5’-TTTCTATCCCCTCCCCCTGG-3’), which was normalized by beta-actin mRNA expression level using specific primers (forward 5’-TCCTTCTGCATCCTGTCGGC-3’ and reverse 5’-CAAGAGATGGCCACGGCTGC-3’).

Transient transfection and dual luciferase assay

A549 cells were transfected with constructed plasmid DNA using lipofectamine (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, 1 × 10⁵ cells/well were seeded onto 12-well plate to obtain 70% to 80% confluence. Cells were transfected with 1 μg of constructed plasmid DNA, 5 ng of Renilla plasmid DNA, 5 μL lipofectamine and grown for 48 hours. HMC-1 cells were transfected with plasmid constructs using a microporator (MP-100, Digital Bio Technology, Seoul, Korea) and Neon transfection system (Invitrogen). Each well of 12-well plate was filled with 1 mL of appropriate medium supplemented with 10% FBS. For each well, 5 × 10⁵ cells mixed with constructed plasmid DNA (1 μg) and Renilla plasmid DNA (5 ng) were suspended in 10 μL of solution R. The mixture then was shocked at 1,650 V/20 mA/plus width 1 in the microporator pipette station containing 3 mL of solution E. Cells were then transferred into 12-well plate and incubated for 24 hours at 37˚C in 5% CO₂ humidified atmosphere. Transfected cells were lysed with passive lysis buffer (Promega). 10 μL of each lysate was applied to a dual-luciferase reporter assay (Promega) using a Clarity Luminescence microplate reader (BioTek, Winooski, VT, USA) following the manufacturer’s protocol. The pGL3-control (Promega) and promoter-less pGL3-basic vectors were used as positive and negative control, respectively. Transfection and luciferase assays were replicated in three independent experiments.

Measurement of serum interleukin 8 by ELISA

Levels of serum interleukin 8 (IL-8) in were measured by using an enzyme-linked immunosorbent assay (ELISA) kit (Endogen, Woburn, MA, USA) according to the manufacturer’s protocol.

Statistical analysis

The chi-square test with one degree of freedom was used to test the frequency of each SNP for significant departure from the Hardy-Weinberg equilibrium in NC group; and p values of less than 0.01 were considered statistically significant [18]. The haplotype block pattern was constructed using Haploview version 4.2 (http://haploview.software.informer.com/4.2/). Haplotypes that had frequency of more than 5% were selected for statistical analysis.

Clinical characteristics were compared between the two study groups using Student t test and chi-square test. Differences in genotype and haplotype frequencies between the study groups were examined by a logistic regression analysis with codominant, dominant and recessive models after accounting for age and sex as covariates. Generalized linear models were applied to compare sputum neutrophil count and serum IL-8 level according to genotypes and haplotypes distribution in the asthma group. The statistical analyses used IBM SPSS version 20 (IBM Co., Armonk, NY, USA) and p values of less than 0.05 were considered statistically significant.

Clinical characteristics of study subjects

Table 3 demonstrates the clinical characteristics of study subjects. Asthma patients were older than NCs (42.4 ± 13.3 vs. 28.6 ± 12.3, p < 0.001). Smoking and atopy rate were significantly higher in the asthma group compared to those of NC group (p = 0.003 and p < 0.001, respectively). Among asthma patients, 17.3% were classified as severe asthma. Mean sputum neutrophil count of the asthma group was 53.6% ± 34.8%.

Table 3.

Clinical characteristics of study subjects

Allele, genotype and haplotype frequency of ATG5 and ATG7 polymorphisms

The distributions of the three SNPs of ATG5 and two SNPs of ATG7 did not depart significantly from the Hardy-Weinberg equilibrium in NC group (p > 0.01 for all SNPs) (Table 1). All the SNPs of each ATG5 and ATG7 were in strong linkage disequilibrium (Fig. 1). Interestingly, the two promoter SNPs of ATG5 gene (–769T>C and –335G>A) were in a complete linkage disequilibrium in our study population; consequently, the statistically analyzed data of –335G>A were shown as the representatives for the two ATG5 promoter SNPs. Three common haplotypes (HT1 [AC], HT2 [GT], HT3 [GC]) for ATG5 –335G>A and 8830C>T; and two common haplotypes (HT1 [AG], HT2 [GC]) for the two SNPs of ATG7 were constructed using Haploview software. The haplotypes that have a frequency more than 5% were selected for analysis. The distribution of genotypes and haplotypes of ATG5 and ATG7 polymorphisms in the two study groups was not significantly different (Tables 4 and 5); and no significant differences in those distributions were noted according to severity of asthma (Tables 6 and 7).

Figure 1.

Linkage disequilibrium plot of ATG5 (A) and ATG7 (B) genetic polymorphisms using Haploview with conf idence bounds color scheme. All single nucleotide polymorphisms were in strong linkage disequilibrium.

Table 4.

Distribution of genotypes and haplotypes of ATG5 genetic polymorphisms into the two study groups

Table 5.

Distribution of genotypes and haplotypes of ATG7 genetic polymorphisms into the two study groups

Table 6.

Distribution of genotypes and haplotypes of ATG5 genetic polymorphisms in asthma group according to asthma severity

Table 7.

Distribution of genotypes and haplotypes of ATG7 genetic polymorphisms in asthma group according to asthma severity

Clinical characteristics according to ATG5 and ATG7 polymorphisms

Fig. 2A shows the comparison of clinical characteristics according to the distributions of ATG5 genotypes in the asthma group. Asthmatics that carried GA/AA genotype at ATG5 –335G>A had significantly higher sputum neutrophil counts compared to those carrying GG genotypes (59.5% ± 33.5% vs. 39.8% ± 34.9%, p = 0.025). Asthmatics carrying CC/CT genotype at ATG5 8830C>T had significantly lower FEV1% predicted values compared to the carriers of TT genotype (83.3% ± 22.1% vs. 92.3% ± 19.2%, p = 0.021). Additionally, the prevalence of asthmatics with FEV1% predicted value < 80% tended to be higher in those carrying ATG5 8830 CC/CT genotype than in those carrying TT genotype (41.0% vs. 22.6%, p = 0.053). Fig. 2B shows that asthmatics carrying ATG5 HT1 [AC] haplotype had a significantly higher sputum neutrophil count compared to non-carriers (58.9% ± 33.6% vs. 39.6% ± 35.5%, p = 0.037); while asthmatic carriers of HT3 [GC] haplotype had a slightly but significantly lower sputum neutrophil count compared to non-carriers (43.5% ± 35.1% vs. 60.1% ± 33.6%, p = 0.049). No associations of HT2 [GT] with clinical parameters were noted.

Figure 2.

Comparison of clinical parameters according to the genotypes of ATG5 and ATG7 polymorphisms in asthmatic patients. (A) Clinical parameters according to the genotypes of ATG5 –335 G>A and 8830C>T. (B) Clinical parameters according to the HT1 [AC] and HT3 [GC] of ATG5 polymorphisms. (C) Serum levels of interleukin (IL) 8 according to ATG7 –100A>G and 25108G>C. A p value was obtained by generalized linear models adjusted for age and sex as covariates.

Regarding ATG7 polymorphisms (Fig. 2C), serum IL-8 levels were significantly higher in asthmatic carriers of GG genotype at –100A>G as well as those carrying CC genotype at 25108G>C compared to the carriers of AA/AG and GG/GC genotypes (137 ± 445 pg/mL vs. 24.2 ± 55.5 pg/mL, p = 0.001; 123 ± 418 pg/mL vs. 24.5 ± 55.9 pg/mL, p = 0.003; respectively).

Transcriptional activity of ATG5 promoter polymorphisms

In order to select the target cell lines to investigate the functional promoter activity of ATG5 promoter polymorphisms, we evaluated the endogenous expression of ATG5 in various human cell lines including lung epithelial cell (A549), bronchial epithelial cell (Beas-2B), promyelocytic leukemia cell (HL-60), leukemic monocyte lymphoma cell (U937) and mast cell (HMC-1). A549 and HMC-1 cell line, known to play role in asthma pathogenesis, were selected based on their high expressions of ATG5 (data not shown).

We constructed two DNA fragments that contained Ht1 [CA] with two minor alleles, and Ht2 [TG] with two major alleles of ATG5 promoter SNPs as described in the Method section for dual luciferase assays. Plasmids with DNA fragments containing major haplotype exhibited significantly higher luciferase activity compared to those containing minor haplotype in both A549 (p < 0.01) (Fig. 3A) and HMC-1 cells (p < 0.001) (Fig. 3B).

Figure 3.

Functional promoter activities of ATG5 polymorphisms. DNA fragments containing Ht1 [CA] or Ht2 [TG] of ATG5 –769T>C and –335G>A were cloned into pGL3-basic vectors. Plasmid constructs were transfected into A549 cells (A) and HMC-1 cells (B). Promoter activities of the DNA fragments were evaluated by dual-luciferase report assays. Transfections and luciferase assays were performed in three independent experiments (total n = 9). Values are presented as mean ± SD; A p value was obtained by one way ANOVA.