Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity and mortality worldwide (Mannino and Buist, 2007). It is characterized by persistent and usually progressive airflow limitation, and is associated with a chronic inflammatory response in the airways to noxious particles or gases such as cigarette smoke (CS). Small airway inflammation and emphysema are the main pathologies of COPD (GOLD, 2016). An increase in the number of inflammatory cells, including cytotoxic T lymphocytes, macrophages and neutrophils, have been observed in patients with COPD, and these cells release inflammatory mediators and enzymes that interact with structural cells in the airways, parenchyma and vasculature (Hogg et al., 2004).

Interleukin-17 (IL-17) is a pro-inflammatory cytokine (Yao et al., 1995) mainly derived from T helper 17 (T_H17) cells (Harrington et al., 2005), which contributes to the pathogenesis of inflammatory disease (Fossiez et al., 1996). The IL-17 family consists of 6 members, IL-17A to F (Kolls and Linden, 2004), of which IL-17A is the most active inducer of inflammation (Zrioual et al., 2009), and five receptors, IL-17RA to IL-17RE (Aggarwal and Gurney, 2002). IL-17A activates pro-inflammatory signaling pathways through binding to the receptors IL-17RA and IL-17RC (Hot and Miossec, 2011). Fibroblasts, endothelial cells and epithelial cells are the major targets of IL-17A (Miossec and Kolls, 2012). A number of studies have demonstrated the contribution of IL-17 to the pathogenesis of COPD (Chen et al., 2011; Di Stefano et al., 2009; Doe et al., 2010; Shan et al., 2012).

Although the pathogenesis of COPD is complicated, inflammation, oxidative stress, and protease-antiprotease imbalance are regarded as a pathogenic triad in COPD (Fischer et al., 2011), and many studies have demonstrated cross talk among them (Gupta et al., 2016; Lee et al., 2015; Kirkham and Barnes, 2013; Rahman and Adcock, 2006; Ungurs et al., 2014). Cigarette smoke itself contains a high concentration of reactive oxygen species (ROS), and therefore there could be a relationship between cigarette smoke and IL-17-induced inflammation. However, this has not been investigated, and the underlying molecular mechanisms are unknown.

In the current study, we show that cigarette smoke extract (CSE) enhances IL-17A-induced IL-8 production via IL-17R up-regulation, and that this is mediated by activation of PI3K-Akt and subsequent inactivation of GSK-3β.

Cells and reagents

Normal human bronchial epithelial cells (BEAS-2B) were maintained in defined keratinocyte-SFM (Gibco by Life Technologies, USA) at 37°C under 5% CO₂. Recombinant human IL-17A (317-ILB-050), mouse monoclonal anti-human IL-17R-allophycocyanin (APC)-conjugated antibody, and an isotype control antibody were purchased from R&D Systems (USA). IL-17A was reconstituted to 100 μg/ml in 4 mM HCl. A rabbit polyclonal anti-IL-17R antibody, to detect the N-terminal extracellular domain of IL-17R, anti-hemagglutinin (HA) and goat polyclonal anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (USA). Rabbit poly-clonal anti-phospho-Akt (Ser473) (p-Akt), anti-phosphoglycogen synthase kinase-3β (Ser9) (p-GSK-3β), anti-phospho-p44/42 MAPK (Thr202/Tyr204) (p-ERK), and rabbit monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38) antibodies were obtained from Cell Signaling (USA). U0126 (a highly selective inhibitor of MEK1 and MEK2), LY294002 (an inhibitor of phosphatidylinositol 3-kinase, PI3K), and SB203580 (a selective inhibitor of p38 MAPK) were obtained from Cell Signaling.

Cigarette smoke extract (CSE)

CSE was prepared as previously described (Lee et al., 2015). Briefly, commercial cigarettes (THIS; 84 mm long, 8 mm diameter; purchased from KT&G, Republic of Korea) were smoked continuously by a bottle system connected to a vacuum machine. Smoke from 20 cigarettes was bubbled into 60 mL phosphate-buffered saline (PBS) (Gibco). Large, insoluble particles in the resulting suspension were removed by filtering the solution through a 0.22-μm filter.

Multiplex bead assay

IL-8 levels in culture supernatants were determined using a commercially available Bio-Plex Pro™ Cytokine Assay Kit (Bio-Rad, USA) according to the manufacturer’s instructions.

Quantitative real-time PCR

Total RNA was isolated using an RNeasy Kit (Qiagen, Germany). cDNA was synthesized from 1 μg of total RNA, using the Reverse Transcription System (Promega, USA). Power SYBR Green (Applied Biosystems, USA) was used for PCR amplification for IL-17RA, IL-17RC and GAPDH. Real-time PCR was performed using the following primers: human IL-17RA (fwd 5′-AGACACTCCAGAACCAATTCC-3′, rev 5′-TCTTAGAGTTGCTCTCCACCA-3′), human IL-17RC (fwd 5′-ACCAGAACCTCTGGCAAGC-3′, rev 5′-GAGCTGTTCACCTG AACACA-3′) and GAPDH (fwd 5′-GAAGGTGAAGGTCG GAGTC-3′, rev 5′-GAAGATGGTGATGGGATTTC-3′).

Protein extraction and western blot analysis

Total cellular extracts were prepared in 1X cell lysis buffer (Cell Signaling). Membrane proteins were isolated using a Membrane Protein Extraction Kit (Thermo Fisher Scientific, USA). Equal amounts of protein were resolved by gradient SDS-polyacrylamide gel electrophoresis (Invitrogen) and transferred to nitrocellulose membranes (GE Healthcare, UK). Membranes were blocked with 5% skim milk blocking buffer for 1 h before being incubated overnight at 4°C with primary antibodies. Membranes were then washed with washing buffer three times and incubated with horseradish peroxidase-conjugated secondary antibodies in blocking buffer for 1 h. After successive washes, membranes were developed using a SuperSignal West Pico Chemiluminescent Kit (Thermo Fisher Scientific).

FACS analysis of the surface membrane receptor, IL-17R

Cells were washed twice in an isotonic PBS buffer supplemented with 0.5% BSA, then resuspended in the same buffer. Cells (1 × 10⁵) were incubated with an APC-conjugated IL-17R antibody or an isotype control antibody for 45 min at 4°C. To remove unreacted IL-17R reagent, cells were washed twice in resuspension buffer. Fluorescence of individual cells was measured using a FACScan flow cytometer (BD, USA).

Transfection of plasmid vectors

A plasmid vector expressing GSK-3β (S9A) (Ser9 to alanine substitution) and a control vector were used at a final concentration of 1 μg. Transfection was performed using a Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer’s specifications. After 48 h, cells were used in experiments.

Statistical analysis

Data were subjected to Student’s t test to determine statistical significance, and a P value of < 0.05 was considered significant.

CSE enhances IL-17A-induced IL-8 production

We first assessed the effect of CSE on IL-17A-induced IL-8 production in BEAS-2B lung epithelial cells. While treatment with IL-17A alone led to a slight increase in IL-8 release, co-treatment with CSE significantly enhanced IL-17A-induced IL-8 production (Fig. 1).

CSE up-regulates IL-17RA/RC expression

IL-17A-induced production of IL-8 is known to be mediated by binding of IL-17A to its receptor, IL-17RA/RC. We therefore investigated whether CSE treatment influences the expression of IL-17RA/RC, and found that CSE increased IL-17RA and IL-17RC mRNA expression (Fig. 2A). To determine whether IL-17R expression was also up-regulated at the membrane level, we isolated membrane proteins from CSE-treated cells and performed western blot analysis using an IL-17R antibody to detect the extracellular domain of IL-17R. Our results revealed that IL-17R in the membrane fraction was increased (Fig. 2B), and this was confirmed by flow cytometry (Fig. 2C). Taken together, these data suggest that CSE up-regulates IL-17R in lung epithelial cells.

Enhanced IL-17A-induced IL-8 production by CSE is dependent on PI3K/Akt activation

We next sought to determine the mechanism by which CSE up-regulates IL-17R expression. Previous reports have shown that CSE can activate both PI3K/Akt and mitogen-activated protein kinase (MAPK) signaling pathways (Kim et al., 2009; Lee et al., 2015). Thus, we sought to investigate whether these pathways are involved in the CSE-induced up-regulation of IL-17R in our system. Our results showed that CSE treatment activated Akt and MAPK (ERK and p38) in lung epithelial cells (Fig. 3A). Furthermore, pretreatment with an inhibitor of the PI3K/Akt pathway (LY294002) significantly suppressed IL-17R expression in the membrane fraction of CSE-treated cells (Fig. 3B). In accordance with these findings, PI3K/Akt inhibition partially suppressed the CSE-induced enhancement of IL-8 production in IL-17A-treated cells (Fig. 3C). These results indicate that enhanced IL-17A-induced IL-8 production by CSE is due, at least in part, to the PI3K/Akt mediated up-regulation of IL-17R.

Increased IL-17R through PI3K/Akt activation is mediated by GSK-3β inactivation

An increase in phosphorylated-Akt was first seen 15 min after CSE treatment, and returned to basal levels after 1 h. GSK-3β is a well-known downstream molecule of Akt. Thus, as Akt inactivates GSK-3β, we investigated whether GSK-3β has a role in the CSE-induced increase in IL-17R. GSK-3β is known to be constitutively active and is inactivated by phosphorylation at serine 9 (Stambolic and Woodgett, 1994). In the present study, we showed that inactive phosphorylated GSK-3β at serine 9 was increased by CSE in a time-dependent manner (Fig. 4A). When Akt activation was inhibited by LY294002 pretreatment, GSK-3β inactivation by CSE was markedly suppressed (Fig. 4B). This suggests that CSE inactivates GSK-3β via activation of the PI3K/Akt pathway. To further evaluate the role of GSK-3β inactivation in CSE-induced IL-17R expression, we determined whether the effect of CSE was blocked by active GSK-3β overexpression. Cells were transiently transfected with either GSK-3β (S9A), a plasmid vector expressing HA-tagged GSK-3β cDNA where serine 9 was substituted for non-phosphorylatable alanine, or a control vector (pcDNA3.1). Because GSK-3β (S9A) cannot be inactivated, it functions as a constitutively active form of GSK-3β. Overexpression of GSK-3β (S9A) was confirmed using immunoblotting against HA (Fig. 4C). CSE-induced IL-17R expression and CSE-induced enhancement of IL-8 production were completely blocked in GSK-3β (S9A)-overexpressing cells, compared to control vector-transfected cells (Figs. 4D and 4E). Taken together, these findings indicate that GSK-3β inactivation via the PI3K/Akt pathway mediates CSE-induced up-regulation of IL-17R.

COPD is characterized by anomalous and persistent inflammation, both local and systemic. Neutrophilic inflammation predominates in the COPD airway wall and lumen. CS induces emphysema and increases lung IL-17A production in mice (Shan et al., 2012). The number of inflammatory cells including neutrophils is attenuated in bronchoalveolar lavage fluid of Il-17a−/− mice when compared to that of wild-type mice. Smoke-induced emphysema is also attenuated in Il-17a−/− mice. In contrast, overexpression of IL-17A in the airways of transgenic mice accelerates the development of emphysema and neutrophilic inflammation after CS exposure (Shan et al., 2012). These findings suggest that IL-17A and IL-17A-induced neutrophilic inflammation are important for COPD pathogenesis.

IL-17 is known to contribute to neutrophilic airway inflammation by the release of neutrophil-mobilizing cytokines from airway cells. IL-8 is the major neutrophil chemotactic factor in the lung (Kunkel et al., 1991). In the present study, and consistent with previous reports, we showed that IL-17A led to the production of IL-8 in human bronchial epithelial cells (Bettelli et al., 2007). Released IL-8 from lung epithelial cells recruits neutrophils into the lung and thus further amplifies chronic inflammation.

T_H17 cells, the major source of IL-17, produce more inflammatory cytokines than Tregs or other T-helper cells (Burgler et al., 2009). T_H17 cells (Vargas-Rojas et al., 2011) and IL-17-positive cells (Di Stefano et al., 2009; Doe et al., 2010) are reported to be increased in the peripheral blood and airways of patients with COPD. The sputum concentration of IL-8 is significantly higher in the patients with COPD than in the control subject (Yamamoto et al., 1997). Together, all of these findings indicate that IL-17A contributes to the pathogenesis of COPD and IL-17A-induced IL-8 production mediates at least partially the airway neutrophilic inflammation.

Moreover, the T_H17 lineage requires optimum concentrations of TGF-β and IL-6 in the surrounding microenvironment (Mangan et al., 2006), both of which are elevated in the serum and sputum of patients with COPD (Mak et al., 2009; Perera et al., 2007). IL-17 has also been linked to increased mucus production via its effects on mucin gene expression in the airway epithelium (Onishi and Gaffen, 2010). These results suggest that IL-17 could be a plausible treatment target for COPD (Cazzola and Matera, 2012).

A recent report showed that CS acts as a T_H17 cell adjuvant by enhancing T_H17 cell differentiation in vitro and in vivo (Chen et al., 2011). However, the existence of cross talk between CS and IL-17A has not been clarified. Our study showed that CSE enhanced IL-17A-induced IL-8 production via up-regulation of IL-17R, revealing that cross talk between CSE and IL-17A increases airway inflammation. Although CS is a well-known cause of COPD, and is widely used in murine models of COPD, we chose to use CSE instead of CS in the current study because we had previously developed a new emphysema model in mice using an intra-tracheal injection of CSE (Lee et al., 2015; 2017).

GSK-3β, one of two isoforms of glycogen synthase kinase 3, is a Ser/Thr kinase (Doble and Woodgett, 2003) that regulates more than 50 substrates and many essential cellular functions, such as glycogen metabolism, cell cycle control, cell differentiation and inflammation (Cortes-Vieyra et al., 2012; Jope and Johnson, 2004). GSK-3β is activated by Tyr216 phosphorylation and is inactivated by Ser9 phosphorylation. Several protein kinases can phosphorylate Ser9, including protein kinase A (PKA), protein kinase B (PKB, Akt), and ribosomal protein S6 kinase (S6K). Of these, the most important GSK-3β inactivator is Akt (Cortes-Vieyra et al., 2012; Jope and Johnson, 2004), which is activated by PI3K (Mahajan and Mahajan, 2012). In the present study, CSE was shown to activate Akt and phosphorylate GSK-3β (Ser9). CSE-induced up-regulation of IL-17R was completely inhibited by a constitutively active form of GSK-3β (S9A), which was not inactivated by Akt. The CSE-induced up-regulation of IL-17R was also suppressed by a chemical inhibitor of PI3K/Akt (LY294002). Other studies have reported that GSK-3β inhibition increases IL-8 production through protein kinase C (PKC)-extracellular signal-related kinase (ERK) signaling (Wang et al., 2006). However, in the current study, the inhibition of ERK did not affect IL-17R expression. GSK-3 has been reported to regulate the activity of numerous factors including c-Jun (Boyle et al., 1991), c-Myc (Gregory et al., 2003), β-catenin (Rubinfeld et al., 1996; Thomas et al., 1999), heat shock factor 1 (Chu et al., 1998), NF-κB (Hoeflich et al., 2000), signal transducers and activators of transcription (STAT) (Ginger et al., 2000), and MAPK/ERK kinase kinase 1 (MEKK1) (Kim et al., 2003). At this time, it is not clear whether the CSE-induced up-regulation of IL-17R is mediated directly by inactive GSK-3β or via the regulation of other signaling molecules.

In conclusion, CSE enhanced the IL-17A-induced IL-8 production by up-regulation of IL-17R, which is caused by CSE-induced activation of the PI3K/Akt pathway and subsequent inactivation of GSK-3β.