A total of 143 patients with COPD (57 non-smokers, 86 smokers) and 75 patients without COPD (45 non-smokers, 30 smokers) were recruited from Hunan Provincial People's Hospital during (Changsha, Hunan Province, China) from March 2014 to September 2016. Patients were excluded from the present study if they exhibited acute and chronic pulmonary diseases, including bronchial asthma, bronchiectasis, interstitial lung disease, heart disease and autoimmune disease. Patients were also excluded if they possessed other systemic disorders, including orthopedic, neurologic or unstable cardiac diseases, which may interfere with results. Patients were recruited into the present study if they had not received glucocorticoid and antibiotics within the last 3 months. A total of 99 male and 44 female patients with COPD and average age of 56.4 years (range, 39–74 years) were recruited. In addition, 43 male and 32 female patients without COPD were recruited (average age, 52.5 years; range, 33–72 years). Lung tissue samples were obtained following lobectomy or pneumonectomy for various medical reasons, including solitary pulmonary nodules, peripheral space-occupying lesions and peripheral lung cancer. COPD was diagnosed according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease (16) as follows: Non-COPD, forced expiratory volume in 1 sec (FEV1)/forced vital capacity (FVC) ≥70% and FEV1 ≥70%; COPD, FEV1/FVC <70% and FEV1 <70%. Patients with a history of any other significant respiratory diseases were excluded. Non-smokers were defined as those who had smoked on average <1 cigarette/day for <1 year or had never smoked. Smokers were defined as patients who had a smoking history of ≥1 year and had smoked on average ≥300 cigarettes/year. None of the patients received corticosteroids prior to surgery. All experiments were approved by the Ethics Committee of Hunan Provincial People's Hospital and informed consent was obtained from all patients prior to specimen collection.

Immunohistochemical (IHC) staining for IL-17A and GDF15 proteins was performed using the labeled streptavidin-biotin method with an LSAB kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Surgical specimens were fixed using 10% neutral-buffered formalin. Following fixation for 24–48 h at room temperature, an initial 3–5 mm thick section was routinely processed, paraffin embedded, deparaffinized and rehydrated. 5-µm sections were then cut, placed on charged slides and dried in an oven for 1 h at 56–60°C. Sections were then stored in the dark at 2–8°C and used for the IHC assay. Sections were then immersed in 0.01 Mcitric buffer (pH 6.0) and preheated to 97°C, for 30 min. The slides were incubated with 3% hydrogen peroxide for 10 min at room temperature, followed by incubation in normal goat serum (cat. no. ab7481; Abcam, Cambridge, UK) for 10 min at room temperature. The slides were incubated with antibodies against IL-17A (ab217359; 1:200) and GDF15 (ab206414; 1:100; both Abcam) at 37°C for 1 h, followed by incubation in Biotin-SP (long spacer) AffiniPure Goat Anti-Rabbit Immunoglobulin G (IgG; 1:2,000; cat. no. 111-065-008; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 10 min at room temperature and subsequently with streptavidin peroxidase for 10 min at room temperature. Diaminobenzidene was employed as the chromogen for 5 min at room temperature. Samples were then counterstained with hematoxylin at room temperature for 30 sec and coverslipped for microscopic examination (50I; light microscope; Nikon Corporation, Tokyo, Japan).

Human small airway epithelial cells (HSAEpiCs) were obtained from ScienCellResearch Laboratories, Inc. (San Diego, CA, USA; 3230) and cultured at 37°C in Small Airway Epithelial Cell Medium (cat. no. 3131; ScienCell Research Laboratories, Inc.) in a humidified atmosphere containing 5% carbon dioxide. To induce GDF15 overexpression, the recombinant expression vector pReceiver-Lv154 which contains the coding sequence of GDF15 (NM_004864) was bought from GeneCopoeia, Inc. (Rockville, MD, USA). Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. L3000015) was used to transfect cells with DNA complexes according to the manufacturer's protocol. Cells were harvested for RNA and protein extraction 72 h following transfection.

CSE was prepared according to a previously published protocol (17). CSE was made from one brand of commercial cigarette (Baisha; Hunan Changsha Tobacco Industrial Co., Ltd., Changsha, China) bubbled through 12.5 ml of bronchial epithelium basal medium (cat. no. CC-3171; Lonza Group Ltd., Basel, Switzerland), which was filtered through a 0.2-mm pore filter. To ensure standardization between experiments and batches of CSE, the absorbance was measured at 320 nm on a spectrophotometer. An optical density of 1 was defined as 100%. CSE was frozen in single use aliquots at −20°C. According to previous reports, 1% CSE is the equivalent of 5 cigarettes/day, 3% is 15 cigarettes/day, 6% is 30 cigarettes/day and 10% is equal to 50 cigarettes/day, which corresponds to human daily exposure to cigarette smoke (18,19). The cytotoxicity of CSE on primary basal cells was measured using a Cytotoxicity Detection kit 1644793001; Roche Diagnostics GmbH, Mannheim, Germany).

Cells were seeded at a density of 1×10⁶ onto 6-well plates and grown in bronchial epithelial growth medium (BEGM; Lonza Group Ltd.) supplemented with a BEGM™ BulletKit™ (cat. no. CC-3170; Lonza Group Ltd.) containing bovine pituitary extract, insulin, hydrocortisone, gentamycin/amphotericin, retinoic acid, transferrin, epinephrine and human epidermal growth factor (Lonza Group Ltd.). The medium was supplemented with heat-inactivated foetal bovine serum (cat. no. 10099141; Gibco; Thermo Fisher Scientific, Inc.; 10% in the growth medium or 1% in starvation medium). At confluence, cells were starved for 24 h (BEGM + 1% FBS), then treated daily with IL-17A 00 ng/ml) and CSE (10%) for 4 days, or treated with varying concentrations of IL7A (10, 25, 50, 100 ng/ml) or CSE (1, 2.5, 5, 10, 15%) for 3 days.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. First strand cDNA was synthesised using Superscript II reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The reverse transcription temperature protocol was 42°C for 50 min, followed by 70°C for 15 min. Real-time PCR reactions were performed using the SYBR-Green Real time PCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) and PCR was performed using an ABI PRISM 7900 HT Sequence Detection System (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 5 min, followed by 95°C for 30 sec, 58°C for 30 sec and 40 cycles of 72°C for 10 sec. All reactions were performed in triplicate. Primers used for the amplification of the indicated genes are listed in Table I. Results were normalised to endogenous GAPDH and quantified using the 2^−∆∆Cq method (20).

Cells were washed with PBS and lysed with RIPA lysis buffer (cat. no. 89900; Invitrogen; Thermo Fisher Scientific) containing a 10% protease inhibitor cocktail (cat. no. 78429; Thermo Fisher Scientific, Inc.) on ice for 30 min. Protein concentrations were determined using a BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Inc.). Aliquots of cell lysates containing 20 µg protein were separated using 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent to blocking with bovine serum albumin (Sigma-Aldrich; Merck KGaA; Darmstadt, Germany) at room temperature for 1 h, membranes were incubated at 4°C overnight with primary antibodies against the following: E-cadherin (1:500; cat. no. ab15148), N-cadherin (1:1,000; cat. no. ab18203), Vimentin (1:1,500; cat. no. ab137321). The anti-E-cadherin, N-cadherin and Vimentin antibodies were obtained from Abcam. Subsequently, membranes were washed with Tris-buffered saline and Tween-20 (Sigma-Aldrich; Merck KGaA) and incubated with horseradish peroxidase-conjugated anti-Rabbit IgG secondary antibodies (1:1,000; cat. no. A21253; Thermo Fisher Scientific, Inc.). Immunoreactive proteins were detected using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.). The relative protein expression was analyzed using Image-Pro plus software 6.0 (Media Cybernetics Inc., Rockville, MD, USA). The expression of β-actin (1:5,000; cat. no. ab227387; Abcam) was used as an internal control to normalize the expressions of other proteins.

Data were analysed using SPSS v.17.0 (SPSS, Inc., Chicago, IL, USA) and are presented as the mean ± standard deviation. The non-parametric Mann Whitney U test was used for comparisons-between two groups. One-way analysis of variance with a post-hoc Tukey's test was used for comparisons between more than two groups. The correlation between gene expression levels was analysed using Pearson's correlation. P<0.05 was considered to indicate a statistically significant difference.

IHC staining and RT-qPCR were performed in 143 samples from patients with COPD and 75 samples from patients without COPD. The results of IHC staining demonstrated that IL-17A and GDF15 were markedly upregulated in lung tissues from patients with COPD compared with non-COPD tissues (Fig. 1A). RT-qPCR revealed that IL-17A and GDF15 expression was significantly increased in patients with COPD compared with patients who did not have COPD (Fig. 1B and C). Furthermore, the expression of IL-17A and GDF15 was significantly increased in smokers compared with non-smokers in the COPD and non-COPD groups (Fig. 1B and C). A significant positive correlation was identified between IL-17A and GDF15 expression (Fig. 1D). These results suggest that upregulated IL-17A and GDF15 expression may contribute to COPD development, particularly in patients with a history of smoking.

A correlation analysis was performed to assess the association between IL-17A, GDF15 and EMT markers [E-cadherin, neural (N)-cadherin and vimentin] in clinical specimens (Fig. 2). IL-17A expression was significantly negatively correlated with E-cadherin (Fig. 2A), whereas it was significantly positively correlated with N-cadherin (Fig. 2C) and vimentin (Fig. 2E). GDF15 expressionwas significantly negatively correlated with E-cadherin (Fig. 2B) and significantly positively correlated with vimentin (Fig. 2F). No significant correlation was observed between GDF15 and N-cadherin expression (Fig. 2D). These results suggest a possible link between increased IL-17A and GDF15 expression and EMT during the development of COPD.

The expression of E-cadherin was significantly downregulated in patients with COPD compared to those without COPD, whereas N-cadherin and vimentin were significantly upregulated (Fig. 3).

To address whether smoking affects GDF15 expression, HSAEpiCs were stimulated with CSE and assessed. The results demonstrated that the expression GDF15 mRNA significantly increased in a dose-dependent manner following CSE exposure for 3 days (Fig. 4A). Additionally, GDF15 expression increased significantly with exposure to 10% CSE in a time-dependent manner, reaching a peak following 3 days of exposure (Fig. 4B). It was also demonstrated that GDF15 expression was significantly upregulated by IL-17A in a dose- and time-dependent manner (Fig. 4C and D), suggesting that GDF15 expression is affected by both cigarette smoke exposure and IL-17A treatment in HSAEpiC cells.

To investigate the role of IL-17A and GDF15 in EMT, HSAEpiC cells were transfected with a GDF15-overexpressing lentivirus and treated with IL-17A (100 ng/ml) for 3 days. The results revealed that HSAEpiC cells lost their honeycomb-like epithelial cell morphology and became spindle shape, which is indicative of EMT (Fig. 5A). RT-qPCR and western blotting were performed to assess the expression of EMT markers and the results demonstrated that E-cadherin was downregulated while N-cadherin and vimentin were upregulated in cells GDF15-overexpressing cells with or without IL-17A treatment compared with control cells (Fig. 5B and C). Furthermore, the combination of IL-17A treatment and GDF15 overexpression had a significantly greater effect on E-cadherin, N-cadherin and vimentin expression compared with GDF15 overexpression alone (Fig. 5B and C). These data suggest that IL-17A, in conjunction with GDF15, is able to induce EMT in HSAEpiC cells.