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. 2014 Apr 24;176(3):461–472. doi: 10.1111/cei.12289

Effects of cigarette smoke on Toll-like receptor (TLR) activation of chronic obstructive pulmonary disease (COPD) macrophages

H J Metcalfe *,✉, S Lea *, D Hughes *, R Khalaf *, K Abbott-Banner †,1, D Singh *
PMCID: PMC4008991  PMID: 24528166

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR-2,-4 and-5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non-typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi-induced TNF-α but not NTHi-induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS-induced TNF-α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS-induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.

Keywords: COPD macrophage, cigarette smoke, TLR, bacteria, immune response

Introduction

Chronic obstructive pulmonary disease (COPD) is a condition where poorly reversible airflow obstruction occurs due to an abnormal inflammatory response to inhaled noxious particles [1]. Cigarette smoking is the most common cause of COPD. There are increased numbers of inflammatory cells in the airways of COPD patients, including macrophages and neutrophils [2]. These innate immune cells normally function to eliminate bacteria by phagocytosis, but in COPD these cells increase the levels of airway inflammation. This is achieved by abnormal secretion of cytokines and chemokines that recruit and activate other immune cells, and also release tissue destructive proteases.

Bacterial proteins activate macrophages through Toll-like receptors (TLRs) such as TLR-2 which binds lipopeptides from non-typeable Haemophilus influenzae (NTHi) [3] and Streptococcus pneumoniae [4], TLR-4 which binds lipopolysaccharide (LPS) and lipooligosaccharide (LOS) from Gram-negative bacteria [5,6] and TLR-5 which binds flagellin from pathogens such as Pseudomonas aeruginosa [5]. Ligand binding to TLRs activates intracellular signalling pathways, such as mitogen-activated protein kinases [MAPK: p38, extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK)] and nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB), which up-regulate the production of inflammatory mediators. The airways of many COPD patients are chronically colonized with bacteria such as non-typeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella caterhallis in the stable state [7]. Furthermore, many acute exacerbations of COPD are due to infections with bacteria [7–9]. TLR stimulation of macrophages may therefore occur in COPD patients during both the stable state and acute exacerbations.

There is evidence that chronic cigarette smoke exposure reduces the proinflammatory response of alveolar macrophages after TLR stimulation. The production of cytokines by COPD alveolar macrophages stimulated with bacterial LPS or NTHi LOS is reduced compared to controls [10,11]. Furthermore, alveolar macrophages from persistent smokers also have a reduced inflammatory response after LPS stimulation, which is associated with decreased activation of NF-κB and p38 MAPK signalling [12]. Cigarette smoke extract (CSE) has been used to study these effects in vitro: CSE reduces macrophage gene expression of many proinflammatory cytokines and chemokines [13]. The exception to this pattern is that CSE increases secretion of the neutrophil chemoattractant interleukin 8 (CXCL8). This occurs through CSE-induced p38 MAPK activation, resulting in post-transcriptional mRNA stabilization [14,15].

In real life, COPD alveolar macrophages may be exposed to both cigarette smoke and bacteria; the majority of previous in-vitro studies have investigated the effects of CSE alone or TLR ligand stimulation alone on COPD macrophage function. Birrell et al. [16] demonstrated that CSE reduced the production of proinflammatory mediators from LPS-stimulated THP-1 cells, with the exception of CXCL8. They confirmed these findings using a small number (n = 5) of human alveolar macrophages. This indicates that smoking has diverse effects on the human macrophage innate immune response, with increased CXCL8 production being an important mechanism for driving neutrophilic airway inflammation under conditions of oxidative stress and bacterial exposure. However, the study by Birrell et al. [16] did not evaluate COPD macrophages. Furthermore, it is important to know whether the effects reported using the TLR-4 ligand LPS are also observed when macrophages are exposed to CSE and other TLR ligands. Importantly, the use of bacteria such as NTHi, which is highly relevant to the pathophysiology of COPD, in conjunction with CSE has not been studied.

The aim of this study was to evaluate the innate immune response of COPD macrophages under physiologically relevant conditions of exposure to both cigarette smoke and bacteria. We have compared the response of COPD and control macrophages to a range of TLR ligands. We also used NTHi as a relevant model of the effects of CSE on macrophage responses to bacteria. The effects of CSE on TLR induced MAPK and NF-κB activation were also evaluated in COPD macrophages, in order to understand how smoking reduces the innate immune response in these cells. The key novelty of this study was that the experiments were performed in COPD macrophages, which are physiologically unique and differ from normal macrophages or cell lines.

Materials and methods

Subjects

Sixty-six patients undergoing surgical resection for suspected or confirmed lung cancer were recruited (see Table 1 for subject demographics). COPD was diagnosed according to current guidelines [1]. Controls with normal lung function and a history of smoking were also recruited. Corticosteroids were used by 43% of the COPD patients. All subjects gave written informed consent. The study was approved by the South Manchester Research Ethics Committee.

Table 1.

Subject demographics.

Smoking status Controls
 COPD
Current Ex Current Ex
Subjects 6 6 29 25
Sex (M/F) 2/4 4/2 13/16 19/6
Age (years) 61·3 ± 7 72·8 ± 11·1 64·8 ± 8·8 71·1 ± 7·5
FEV1 (l) 2 ± 0·6 2·4 ± 0·5 1·8 ± 0·6 1·7 ± 0·6
FEV1 % predicted 73·2 ± 13·4 97·5 ± 18·0 69·7 ± 14·8 70·2 ± 19·9
FVC 2·8 ± 1·0 3·3 ± 0·4 3·2 ± 1·1 3·2 ± 0·8
FEV1 : FVC ratio 73·8 ± 10·3 74·4 ± 6·4 58·0 ± 8·9 55·8 ± 11·9
Smoking history (pack year) 56·6 ± 25·9 30·8 ± 13·6 53·7 ± 41·4 44·7 ± 27·6
ICS users 0 0 10 12
OCS users 0 0 1† 1
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†

One current smoker chronic obstructive pulmonary disease (COPD) patient was on both ICS and OCS. Data presented as mean ± standard deviation. M = male; F = female; FEV1 = forced expiratory volume in 1 second; FVC = forced vital capacity; ICS = inhaled corticosteroid; OCS = oral corticosteroid.

Preparation of CSE

CSE was prepared as described previously [17] by bubbling two 3R4F Kentucky cigarettes (University of Kentucky, Lexington, KY, USA) through 25 ml RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 1% penicillin/streptomycin (Sigma, Poole, UK) and 1% L-glutamine (Invitrogen) using a pump (15 ml/min). Afterwards, 10% fetal calf serum (FCS; Invitrogen) was added and the CSE was filtered (22 μm filter; Millipore, Watford, UK). CSE concentration was determined by measuring optical density (OD) using the 320 nm wavelength on the spectrophotometer (Eppendorf, Stevenage, UK). CSE was adjusted to the desired OD in culture media.

Bacterial cell culture

NTHi strain R2846 (kindly provided by Dr Nicola High, University of Manchester, Oxford Road, Manchester) was streaked on chocolate agar plates (E&O Laboratories, Bonnybridge, UK) and cultured at 37°C for 16 h. Individual colonies were cultured for 16 h (37°C at 150 rpm, 311DS Labnet orbital incubator) in brain heart infusion (BHI) broth (Sigma) supplemented with 1 mg/ml β-nicotinamide adenine dinucleotide (NAD; Sigma), 1 mg/ml L-histidine (Invitrogen) and 1 mg/ml Hemin (BioXtra) (Sigma), as described previously [18]. Bacterial density was adjusted to 1·2 OD [20 × 109 colony-forming units (CFU)/ml], using the 600 nm wavelength in the spectrophotometer (BMG Labtech, Aylesbury, UK), and diluted to give an end-point multiplicity of infection (MOI) of 100:1.

Alveolar macrophage culture

Alveolar macrophages were isolated from lung samples as described previously [19]. Macrophages (1 × 106 ml−1) were adhered overnight in RPMI-1640 supplemented with 10% FCS, 1% penicillin/streptomycin and 1% L-glutamine (37°C, 5% CO2) and washed before each treatment. Macrophages were pretreated with and without 0·1 OD CSE for 2 h prior to stimulation with 0·001 μg/ml phase I flagellin (FliC) derived from Salmonella typhimurium (Enzo Life Sciences, Exeter, UK), 0·1 μg/ml ultra-pure Escherichia coli O111:B4 LPS (UPLPS; Invivogen, San Diego, CA, USA), 0·1 μg/ml synthetic bacterial lipoprotein Pam3CSK4 (PAM; Invivogen) or NTHi (MOI 100:1), for time-periods stated in the results. Cytokine/chemokine levels and lactate dehydrogenase (LDH) activity were analysed in 96-well plate alveolar macrophage cultures after 24 h; 1% Triton X (Sigma) was used as a positive cell death control. Twenty-four-well plate cultures were used to analyse CXCL8 and tumour necrosis factor (TNF)-α gene expression following UPLPS stimulation, and activation of p38, ERK and the NF-κB subunit p65 after UPLPS stimulation.

Cytokine/chemokine protein assays

CXCL8 and TNF-α protein levels were analysed by enzyme-linked immunosorbent assay (ELISA) as per the manufacturer's instructions (R&D Systems, Abingdon, UK). Interleukin (IL)-6, IL-10 and regulated upon activation, normal T cell expressed and secreted (RANTES) were analysed using multi-plex Meso Scale Discovery kits (Rockville, MD, USA).

LDH assay

The effect of CSE on alveolar macrophage membrane integrity was assessed by analysing the release of cytoplasmic LDH in the supernatant using the LDH-based in-vitro toxicology assay kit (Sigma). Briefly, 50 μl of culture supernatant was placed in each well of a flat-bottomed 96-well plate with 100 μl lactate dehydrogenase assay mixture. Plates were incubated in the dark for 20 min. Reactions were stopped by adding 15 μl 1 M HCl to each well. Absorbance was measured using the 490 nm wavelength on the spectrophotometer (BMG Labtech).

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay

DNA fragmentation was analysed in chamber slides (Thermo Scientific, Runcorn, UK) using the In Situ Cell Death Detection Kit fluorescein kit (Roche, Welwyn Garden City, UK). Briefly, 50 μl enzyme solution (1/10 in label solution) was added to each chamber of the chamber slide, excluding the negative TUNEL control (macrophages conditioned with 0·2 OD CSE and treated with 50 μl label solution). The chamber slide was protected from light and incubated for 1 h at 37°C. All cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) [1/50 000 in phosphate-buffered saline (PBS) for 5 min in the dark], analysed by microscope (Nikon ECLIPSE 80i), and images were taken using Image-Pro Plus (MediaCybernetics, Marlow, UK). Illustrations of TUNEL staining can be found in the Supporting information, Fig. S1.

Quantitative polymerase chain reaction (qPCR) analysis

As described previously [15], cell pellets were lysed in Trizol (Invitrogen, Paisley, UK) for RNA extraction and PCR analysis for CXCL8, TNF-α, TLR-2, TLR-4 and TLR-5. Total RNA was purified from cell lysates using the RNeasy Mini Kit (Qiagen, Crawley, UK), according to the manufacturer's instructions. DNA contamination was prevented by on-column addition of DNase (Qiagen), according to the manufacturer's instructions. TaqMan reverse transcription–PCR (RT–PCR) was performed on RNA using the Verso™ two-step quantitative RT (qRT)–PCR kit (Thermofisher, Epsom, Surrey, UK), where total RNA was converted to complementary DNA (cDNA). Fifty ng of the cDNA was used in 25-μl reactions containing ABsolute Blue qPCR mix and TaqMan gene expression assay primers for CXCL8, TNF-α, TLR-2, TLR-4 and TLR-5 and for endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Warrington, UK). Thermal cycling was carried out on a Stratagene MX3005P (Agilent Technologies, West Lothian, UK). Amplification conditions were 95°C for 15 min and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative fold induction of gene expression above unstimulated time-matched controls was determined using the ΔΔCt method normalizing to the endogenous GAPDH control.

Western blot analysis

Western blots were run as described previously [20]. All primary antibodies were raised in rabbit: phospho-p38 MAPK (Th180/Tyr182) antibody (Cell Signalling, Hitchin, UK), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody (Cell Signalling), phospho-NF-κB p65 (Ser536) antibody (Cell Signalling) and β-actin antibody (Abcam) as loading control. Horseradish peroxidase-conjugated goat anti-rabbit antibodies (DakoCytomation, San Diego, CA, USA) were used for the secondary antibody. All antibodies were diluted 1/1000.

Statistics

ELISA, multiplex and cytokine gene expression data were not normally distributed and were compared between subject groups using Mann–Whitney U-tests. Data were compared within subject groups using Wilcoxon's paired t-tests. LDH assay and TLR gene expression data were normally distributed and analysed using the repeated-measures one-way analysis of variance (anova) with Bonferroni's post-hoc test. All data were analysed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA) and InStat version 3·06. P < 0·05 was considered significant.

Results

Optimization of CSE concentration

The concentration response curve to CSE in COPD alveolar macrophages was determined initially by measuring CXCL8 production, which is known to be up-regulated by alveolar macrophages following CSE exposure [14,15]. Alveolar macrophages from nine COPD patients exposed to different concentrations of CSE showed significantly increased CXCL8 secretion at OD from 0·03 to 0·1 (Fig. 1a). There was no evidence of cytotoxicity at these OD, as there was no change in LDH secretion (Fig. 1b) or DNA integrity (Fig. 1c); 0·2 OD CSE caused an increase in LDH levels and DNA fragmentation and did not increase CXCL8 secretion. The 0·1 OD CSE concentration was used for further experiments, as it caused the greatest increase in CXCL8 levels without causing cell toxicity. This CSE concentration was found to induce phosphorylation of p38, but not ERK or p65 (Fig. 2).

Figure 1.

Figure 1

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Shows 0·1 optical density (OD) cigarette smoke extract (CSE)-induced significant interleukin 8 (CXCL8) without cytotoxic effects. Alveolar macrophages from nine chronic obstructive pulmonary disease (COPD) patients (two current and seven ex-smokers) were treated with and without CSE (0·001–0·2 OD) for 2 h followed by an additional 24 h with CSE-free medium. Culture supernatants were analysed for CXCL8 (a, n = 9) and lactate dehydrogenase (LDH) activity (b, n = 9). DNA fragmentation was analysed using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (c, n = 3). Data show median and range of CXCL8 levels (a) and mean ± standard error of the mean (s.e.m.) for LDH activity (b) and TUNEL staining (c). **, *** = significant increase in CXCL8 compared to negative control (P < 0·01, 0·001, respectively). **, # = significant increase in LDH activity compared to negative control (P < 0·01, 0·05, respectively).

Figure 2.

Figure 2

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Shows 0·1 optical density (OD) cigarette smoke extract (CSE)-induced p38 activation. Alveolar macrophages from chronic obstructive pulmonary disease (COPD) patients were exposed to either 0·1 OD CSE or medium over a 90-min time–course and protein extracts were analysed for phosphorylation of p38 (pp38, n = 3), extracellular signal regulated kinase (pERK, n = 2) and p65 (pp65, n = 2) by Western blot, using β-actin as a loading control.

Optimization of TLR responses

TLR ligand concentration response curves were investigated using COPD alveolar macrophages (six COPD patients; see online Supporting information, Fig. S2). Optimal cytokine responses were observed with the following TLR ligand concentrations, which were used in subsequent experiments: 0·001 μg/ml FliC, 0·1 μg/ml UPLPS and 0·1 μg/ml PAM.

CSE effects on TLR stimulation

Cytokine secretion

Alveolar macrophages from 12 controls and 12 COPD patients (six current and six ex-smokers in both groups) were exposed to 0·1 OD CSE for 2 h, and then stimulated for 24 h with FliC, UPLPS, PAM or left unstimulated (Figs 3 and 4). All the TLR ligands significantly increased the secretion of TNF-α, IL-6, IL-10, RANTES and CXCL8 compared to the unstimulated control (P < 0·05 for all comparisons). There was no difference in cytokine production between current and ex-smokers (P > 0·05 for comparisons) within the control and COPD groups for CXCL8 and TNF-α.

Figure 3.

Figure 3

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Cigarette smoke extract (CSE) exposure had no effect on Toll-like receptor (TLR)-induced interleukin 8 (CXCL8) but suppressed TLR-induced tumour necrosis factor (TNF)-α. Alveolar macrophages from 12 controls (a,c) and 12 chronic obstructive pulmonary disease (COPD) patients (b,d) (six current and six ex-smokers in both groups) were pretreated for 2 h with either 0·1 optical density (OD) CSE or medium and stimulated for an additional 24 h with 0·001 μg/ml phase I flagellin (FliC), 0·1 μg/ml ultra-pure Escherichia coli lipopolysaccharide (UPLPS), 0·1 μg/ml Pam3CSK4 (PAM) or left unstimulated (Unstim). Supernatants were analysed for CXCL8 and TNF-α. Data show median and range for CXCL8 (a,b) and TNF-α (c,d). *, **, *** = significant difference compared to matched medium control (P < 0·05, P < 0·01 and P < 0·001, respectively).

Figure 4.

Figure 4

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Cigarette smoke extract (CSE) exposure suppressed Toll-like receptor (TLR)induced interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES). Alveolar macrophages from eight controls (three current and five ex-smokers) or seven chronic obstructive pulmonary disease (COPD) patients (four current and three ex-smokers) were pretreated for 2 h with either 0·1 OD CSE or medium and stimulated for an additional 24 h with 0·001 μg/ml phase I flagellin (FliC), 0·1 μg/ml ultra-pure Escherichia coli lipopolysaccharide (UPLPS), 0·1 μg/ml am3CSK4 (PAM) or left unstimulated (Unstim). Supernatants were analysed for IL-6, IL-10 and RANTES using MesoScale Discovery (MSD) kits. Data show median and range for IL-6 (a,b), IL-10 (c,d) and RANTES (e,f). *, ** = significant difference compared to matched medium control (P < 0·05 and P < 0·01, respectively).

CSE alone, without subsequent TLR stimulation, increased the secretion of CXCL8 protein within both the COPD and control groups. In contrast, CSE alone had no effect on the secretion of TNF-α, IL-6 or IL-10, and decreased RANTES secretion in the COPD group but not in the controls.

CSE exposure significantly reduced TLR-stimulated TNF-α, IL-6, IL-10 and RANTES secretion within both the COPD and control groups. However, CSE exposure did not change TLR-stimulated CXCL8 levels in either group.

TNF-α, IL-6, RANTES and CXCL8 levels were not different between the COPD and control groups in any of the experimental conditions, both with and without CSE exposure (P > 0·05 for all comparisons). However, unstimulated and CSE-exposed COPD alveolar macrophages had higher IL-10 levels compared to controls (P < 0·05 for both comparisons). As there were few differences between the results from COPD and controls, all further experiments were then performed using COPD macrophages only.

CXCL8 and TNF-α gene expression

Alveolar macrophages from nine COPD patients were pretreated for 2 h with and without 0·1 OD CSE, and then stimulated for 6 or 24 h with 0·1 μg/ml UPLPS or left unstimulated (Fig. 5). CXCL8 gene expression was increased by UPLPS at 6 and 24 h. CSE alone also caused an increase in CXCL8 gene expression at both 6 and 24 h, although this was lower than the effect of UPLPS. CSE exposure followed by UPLPS stimulation resulted in similar CXCL8 gene expression compared to UPLPS stimulation alone.

Figure 5.

Figure 5

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The effect of cigarette smoke extract (CSE) on ultra-pure Escherichia coli lipopolysaccharide (UPLPS)-induced interleukin 8 (CXCL8) and tumour necrosis factor (TNF)-α gene expression. Alveolar macrophages from nine chronic obstructive pulmonary disease (COPD) patients (eight current and one ex-smoker) were pretreated for 2 h with either 0·1 optical density (OD) CSE or medium and stimulated for an additional 6 h (a,c) or 24 h (b,d) with 0·1 μg/ml UPLPS or left unstimulated (Unstim). CXCL8 and TNF-α gene expression was analysed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH). Data show median and range for CXCL8 (a,b) and TNF-α (c,d). *, ** = significant difference compared to matched medium control (P < 0·05 and P < 0·01, respectively).

CSE alone did not change TNF-α gene expression. UPLPS increased TNF-α gene expression at 6 h, which returned to unstimulated levels by 24 h (P > 0·05). CSE reduced UPLPS stimulated TNF-α gene expression levels at 6 h.

p38, ERK and p65 activation

Having observed that CSE inhibits TLR mediated production of certain cytokines (TNF-α, IL-6, IL-10 and RANTES), we investigated whether CSE suppressed p38, ERK and p65 signalling after TLR stimulation. Alveolar macrophages from six COPD patients were pretreated for 2 h with and without 0·1 OD CSE, then stimulated for 30 min with 0·1 μg/ml UPLPS or left unstimulated. Representative Western blots are shown in Fig. 6, with quantitative densitometry. Western blot analysis showed phosphorylation of p38, ERK and p65 after UPLPS stimulation alone. CSE pretreatment significantly reduced the phosphorylation of all of these proteins.

Figure 6.

Figure 6

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Cigarette smoke extract (CSE) exposure dampened ultra-pure Escherichia coli lipopolysaccharide (UPLPS)-induced activation of p38, extracellular signal regulated kinase (ERK) and p65. Alveolar macrophages from six chronic obstructive pulmonary disease (COPD) patients (two current and four ex-smokers) were pretreated for 2 h with either 0·1 optical density (OD) CSE or medium and stimulated for an additional 30 min with 0·1 μg/ml UPLPS or left unstimulated (Unstim). Phosphorylation of p38 (pp38), ERK (pERK) and p65 (pp65) was analysed by Western blot. Band intensities were calculated by normalizing to β-actin. Data show median and range for pp38 (a), pERK (b) or pp65 (c). * = significant difference compared to matched medium control (P < 0·05).

CSE effects on TLR gene expression

Alveolar macrophages from seven COPD patients were incubated with 0·1 OD CSE for 48 h (Fig. 7). There was significant down-regulation of TLR-5 gene expression at 4 and 6 h, but CSE had no effect on TLR-4 (anova P = 0·74) and TLR-2 (anova P = 0·13) gene expression.

Figure 7.

Figure 7

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Cigarette smoke extract (CSE)-induced significant down-regulation of Toll-like receptor (TLR)-5 gene expression without affecting TLR-4/-2 gene expression. Alveolar macrophages from seven chronic obstructive pulmonary disease (COPD) patients (four current and three ex-smokers) were exposed to 0·1 optical density (OD) CSE over a 48-h time–course. TLR-5, TLR-4 and TLR-2 gene expression was analysed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH). Expression levels are calculated relative to the time-matched unstimulated controls. Dot-plots show means and standard error of the mean for TLR-5 (a), TLR-4 (b) and TLR-2 (c). *** = significant difference compared to medium control (P < 0·001).

NTHi infection of CSE exposed COPD alveolar macrophages

Alveolar macrophages from six COPD patients were pretreated for 2 h with either 0·1 OD CSE or medium, and then cultured for an additional 24 h with 0·1 μg/ml UPLPS, 100:1 MOI NTHi or left unstimulated (Fig. 8). NTHi caused greater secretion of TNF-α compared to UPLPS (P = 0·03), but there was no difference between groups for CXCL8. CSE (0·1 OD) had no effect on CXCL8 production after infection with NTHi or stimulation with UPLPS, but significantly reduced TNF-α production after infection with NTHi or stimulation with UPLPS.

Figure 8.

Figure 8

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The effect of cigarette smoke extract (CSE) on ultra-pure Escherichia coli lipopolysaccharide (UPLPS) and non-typeable Haemophilus influenzae (NTHi)-induced interleukin 8 (CXCL8) and tumour necrosis factor (TNF)-α. Alveolar macrophages from six chronic obstructive pulmonary disease (COPD) patients (three current and three ex-smokers) were pretreated for 2 h with either 0·1 OD CSE or medium and stimulated for an additional 24 h with 0·1 μg/ml ultra-pure Escherichia coli lipopolysaccharide (UPLPS), NTHi [100 : 1 multiplicity of infection (MOI)] or left unstimulated (Unstim). Supernatants were collected and analysed for CXCL8 and TNF-α secretion by enzyme-linked immunosorbent assay (ELISA). Data show median and range for CXCL8 (a) and TNF-α (b). * = significant difference compared to matched medium control (P < 0·05).

Discussion

We have used COPD macrophages to evaluate the effects of CSE on the innate immune response to a range of TLR ligands and NTHi. CSE suppressed TLR-induced cytokine production in COPD and control alveolar macrophages, including NTHi-induced cytokine production in COPD alveolar macrophages. This effect was associated with a reduction in TLR-induced MAPK and NF-κB activation. However, CSE had no effect on NTHi-and TLR-induced CXCL8 production.

Studies using COPD macrophages exposed to CSE alone have demonstrated that CXCL8 secretion is increased while the expression of other proinflammatory mediators is decreased [13,17,21]. The key novel aspect of this study is that we have used COPD macrophages exposed to both CSE and a range of TLR ligands, in addition to NTHi. The use of NTHi resembles the real-life situation where COPD alveolar macrophages are commonly exposed to both cigarette smoke and this particular bacterium. Our findings demonstrate that NTHi can cause neutrophilic inflammation in the airways of COPD patients through persistent CXCL8 production, despite cigarette smoke-related suppression of other components of the macrophage innate immune response.

CXCL8 is a potent neutrophil chemoattractant which is secreted into the airways of COPD patients at higher levels compared to healthy controls [22–24]. CXCL8 is produced by different cells in the airways, including macrophages, neutrophils and epithelial cells. Bronchoscopy studies have reported that COPD patients with bacterial airway colonization have increased CXCL8 levels and neutrophil numbers in the airways [25,26]. Our results are compatible with these findings, as we have shown that a range of TLR ligands and NHTi up-regulate CXCL8 production from COPD alveolar macrophages, despite suppression of the secretion of other cytokines by CSE, supporting the association between bacterial colonization and CXCL8 production.

The measurement of TNF-α and CXCL8 gene expression offers insights into the differences observed between these cytokines with CSE exposure. CXCL8 gene expression differed from TNF-α in two ways; CXCL8 showed prolonged up-regulation after TLR stimulation, in keeping with data shown previously by our group [27], and only CXCL8 gene expression was up-regulated by CSE. The secretion of CXCL8 is controlled by NF-κB, p38 MAPK and activator protein-1 (AP-1) [28], with p38 MAPK playing an important role in mRNA stabilization. We show here that CSE activates p38 MAPK in COPD alveolar macrophages, in agreement with previous work also showing that CSE-induced activation of this MAPK increased CXCL8 mRNA stabilization [15]. The signal transduction mechanisms controlling TNF-α and CXCL8 gene expression in COPD alveolar macrophages appear to be different, with CSE activation of p38 MAPK playing a selective role in up-regulation of the transcription of CXCL8 but not TNF-α shown here, and CXCL8 mRNA stabilization shown previously [15]. The potential role of p38 MAPK in the pathophysiology of COPD is well recognized, with increased expression of phospho-p38 MAPK in alveolar macrophages of smokers compared to non-smokers, with a further increase in COPD patients compared to smokers [29].

We observed that CSE reduced TLR-stimulated ERK, p38 and p65 activation; these signalling pathways are involved in the production of proinflammatory cytokines. It has also been shown previously that alveolar macrophages from smokers have reduced p38 and NF-κB signalling after LPS stimulation compared to non-smokers [12]. The lack of CSE suppression of TLR-stimulated CXCL8 production is likely to be caused by the initial activation of p38 MAPK by CSE.

After CSE exposure, the subsequent TLR stimulation did not cause another peak of p38 MAPK activation. This is probably because homeostatic regulatory mechanisms have already been activated to control p38 MAPK signalling. Nevertheless, the initial CSE activation of p38 MAPK, with the subsequent effect on CXCL8 mRNA stabilization, is likely to be the cause of maintained CXCL8 secretion in the experiments where CSE exposure was followed by TLR stimulation.

It has been shown previously that p38 MAPK inhibitors reduce CXCL8 secretion from LPS-stimulated COPD alveolar macrophages [19,20]. These drugs also reduce CXCL8 mRNA stabilization in CSE-exposed alveolar macrophages [15]. We would therefore expect p38 MAPK inhibitors to also reduce CXCL8 production from COPD alveolar macrophages exposed to CSE and then LPS.

Previous studies have shown a variety of results concerning the effects of CSE on NF-κB and MAPK signalling, with reports that CSE is capable of activating ERK, JNK and p65 [30–34], as well as suppressing these pathways [35,36]. However, these previous studies did not use COPD alveolar macrophages, and our results concerning these signalling pathways are in agreement with previous studies that have used COPD alveolar macrophages [14,15].

NTHi caused more TNF-α secretion than LPS, which may be attributed to multiple TLR receptor stimulation due to the different microbial-associated molecular patterns expressed by NTHi. Interestingly, the effects of NTHi and LPS on CXCL8 secretion were similar to each other, further underscoring that differences exist in signalling mechanisms driving TNF-α and CXCL8 secretion in COPD alveolar macrophages.

IL-10 is a regulatory anti-inflammatory cytokine [37] which appears to be suppressed in the airways of smokers and COPD patients [38]. In this study, we observed that IL-10 secretion in unstimulated and CSE-exposed cells was higher in COPD alveolar macrophages compared to controls, consistent with the switching of COPD alveolar macrophages towards the M2-like phenotype [39], which is anti-inflammatory and involved in tissue repair [40–42]. However, CSE reduced IL-10 secretion in TLR-stimulated cells from both groups; this suggests that CSE causes a suppression of this anti-inflammatory mechanism under conditions of bacterial exposure.

Apart from the difference between groups for IL-10, we did not observe any other differences in cytokine production between the COPD and control groups. Previous studies have shown that chronic smoking reduces LPS-stimulated secretion of cytokines from alveolar macrophages, and that COPD macrophages have a reduced cytokine response compared to smokers [10–12]. Perhaps the failure to show a difference between groups in the current study was due to the mild to moderate nature of the COPD patients; a reduced LPS response may have been observed in more severe patients. Nevertheless, our observations in this study, that acute CSE exposure dampens certain components of the innate immune response, reflects these previous studies where chronic smoking has a similar effect.

The CSE concentration selected in this study (0·1 OD) did not cause a reduction in membrane integrity or an increase in DNA fragmentation in COPD alveolar macrophages. These mechanisms occur during apoptosis or necrosis, and we were careful to select a CSE concentration that did not have these effects. Interestingly, CSE selectively down-regulated TLR-5 gene expression without affecting TLR-4 or TLR-2 gene expression. The reduction in flagellin-stimulated cytokine production caused by CSE might be due partially to a reduction in TLR-5 expression, but this was not the case for TLR-2 or TLR-4. It has been shown that activation of PI3K by TLR-5 limits proinflammatory gene expression by suppressing MAPK activation in mouse epithelial cells [43]. The inhibition of TLR-5-induced PI3K leads to prolonged activation of MAPK (p38 and ERK1/2) and enhancement of CXCL8 gene expression [43]. The reduction of TLR-5 by CSE observed here may play a role in the sustained production of CXCL8 by removing the negative regulation of MAPK p38 provided by PI3K.

The experiments reported in this study used a single exposure to LPS or NTHi to stimulate cytokine production through TLR signalling; this model mimics the macrophage response to acute bacterial exposure. A reduced macrophage response to LPS occurs after chronic exposure; this is known as LPS tolerance [44], and is likely to occur during chronic bacterial exposure. It would be of interest to investigate the effect of CSE on the immune response of LPS tolerized macrophages.

We isolated alveolar macrophages by plate adherence, which is a standard methodology [12,16,45]. However, it is known that plate adherence can alter macrophage behaviour, for example by increasing CXCL8 production [46]. This response may be regarded as resembling the behaviour of macrophages within tissue, or it may be regarded as somewhat non-physiological as it is induced by plastic. Regardless, it is a practical necessity for in-vitro studies of macrophage behaviour, and results in a change in the biological characteristics of these cells. Furthermore, such an effect is equal in all conditions, so is unlikely to have altered our major conclusions.

It has been demonstrated previously that acute cigarette smoke exposure suppresses macrophage phagocytosis [35,47], and that phagocytosis is impaired in COPD macrophages compared to controls [48]. Defective phagocytosis allows bacterial persistence in the airways, leading to increased TLR stimulation. Cigarette smoke exposure therefore alters the nature of the interaction of COPD alveolar macrophages with bacteria by reducing their ability to phagocytose and, as we show here, altering their innate immune response to these pathogens.

In conclusion, we have shown that the innate immune response of COPD alveolar macrophages in response to simultaneous cigarette smoke and bacterial exposure is complex, showing down-regulation of some proinflammatory functions but up-regulation of CXCL8 production. p38 MAPK appears to play a key role in this differential regulation, as it is activated by CSE alone with an associated increase in CXCL8 protein and gene expression.

Acknowledgments

This research was funded by Novartis. We would also like to acknowledge Dr Nicola High (University of Manchester, Oxford Road, Manchester) for providing the NTHi strain R2846.

Disclosures

D. S. has received sponsorship to attend international meetings, honoraria for lecturing or attending advisory boards and research grants from various pharmaceutical companies including Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, CIPLA, Forest, Genetech, GlaxoSmithKline, Merck, Novartis and Pfizer. K. A.-B. was working in Novartis at the time of the study but is now employed by Verona Pharma plc.

Author contributions

H. M. performed the experiments and drafted the paper; D. S. is the principle investigator who reviewed and edited the paper; K. A.-B. collaborated in this study and reviewed the paper; S. L. provided intellectual support and edited the paper; D. H. conducted preliminary investigations (unpublished research) which led to these studies; and R. K. assisted in the NTHi experiments.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig S1

Representative images of composites from the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Alveolar macrophages from three chronic obstructive pulmonary disease (COPD) patients were treated with and without cigarette smoke extract (CSE) [0·1/0·2 optical density (OD)] for 2 h followed by an additional 24 h with CSE-free medium; 1% Triton X was used as a positive cell death control and the 0·2 OD CSE condition was used for the negative TUNEL control [0·2 OD (−)]. Blue fluorescence illustrates 4',6-diamidino-2-phenylindole (DAPI) labelling of DNA in the nucleus and green fluorescence illustrates TUNEL labelling of DNA fragmentation.

cei0176-0461-sd1.tif (423.8KB, tif)
Fig S2

Dose-dependent time–course of Toll-like receptor (TLR) ligand-induced interleukin 8 (CXCL8)/tumour necrosis factor (TNF)-α secretion from chronic obstructive pulmonary disease (COPD) alveolar macrophages. Alveolar macrophages from six COPD patients (two current and seven ex-smokers) were treated with varying doses of phase I flagellin (FliC) (a,d), ultra-pure Escherichia coli lipopolysaccharide (UPLPS) (b,e) and Pam3CSK4 (PAM) (c,f) during 48 h. Collected supernatants were analysed for CXCL8 (a–c) and TNF-α (d–f) by enzyme-linked immunosorbent assay (ELISA). Data sets from each time-point were analysed using the Friedman test and Dunn's multiple comparison test. *, **, *** = significant difference compared to matched medium control (P < 0·05, P < 0·01 and P < 0·001, respectively).

cei0176-0461-sd2.tif (2.4MB, tif)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig S1

Representative images of composites from the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Alveolar macrophages from three chronic obstructive pulmonary disease (COPD) patients were treated with and without cigarette smoke extract (CSE) [0·1/0·2 optical density (OD)] for 2 h followed by an additional 24 h with CSE-free medium; 1% Triton X was used as a positive cell death control and the 0·2 OD CSE condition was used for the negative TUNEL control [0·2 OD (−)]. Blue fluorescence illustrates 4',6-diamidino-2-phenylindole (DAPI) labelling of DNA in the nucleus and green fluorescence illustrates TUNEL labelling of DNA fragmentation.

cei0176-0461-sd1.tif (423.8KB, tif)
Fig S2

Dose-dependent time–course of Toll-like receptor (TLR) ligand-induced interleukin 8 (CXCL8)/tumour necrosis factor (TNF)-α secretion from chronic obstructive pulmonary disease (COPD) alveolar macrophages. Alveolar macrophages from six COPD patients (two current and seven ex-smokers) were treated with varying doses of phase I flagellin (FliC) (a,d), ultra-pure Escherichia coli lipopolysaccharide (UPLPS) (b,e) and Pam3CSK4 (PAM) (c,f) during 48 h. Collected supernatants were analysed for CXCL8 (a–c) and TNF-α (d–f) by enzyme-linked immunosorbent assay (ELISA). Data sets from each time-point were analysed using the Friedman test and Dunn's multiple comparison test. *, **, *** = significant difference compared to matched medium control (P < 0·05, P < 0·01 and P < 0·001, respectively).

cei0176-0461-sd2.tif (2.4MB, tif)

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