Introduction
The lung represents the largest epithelial surface in the body and the respiratory epithelial cell represents the body's first interaction with airborne pathogens. As well as providing a physical barrier to entry of micro-organisms, the epithelium is increasingly recognised to play an important role in innate immunity, and can respond to potential pathogens by releasing a variety of effector molecules of the inflammatory response along with anti-microbial peptides [
1,
2].
TLR4 is critically important in signalling the inflammatory response to Gram-negative bacteria through recognition of LPS, regulating the inducible expression of many cytokines, chemokines, adhesion molecules and acute phase proteins. We have previously shown that LPS signalling via TLR4 induces production of the anti-microbial peptide human beta-defensin 2 (HBD2) [
3], which has a broad spectrum of antimicrobial activity, particularly against Gram-negative bacteria, including
Escherichia coli and
Pseudomonas aeruginosa and the yeast
Candida albicans [
4].
Chronic Obstructive Pulmonary Disease (COPD) is a condition characterised by progressive airflow limitation punctuated by exacerbations, associated with airway inflammation [
5,
6]. The role of bacteria in the pathogenesis and acceleration of COPD remains the subject of some debate, but increasing evidence in recent years supports the importance of bacteria in this disease, as a stimulus to chronic inflammation and a cause of exacerbations [
7]. Modulation of TLR4 expression in respiratory epithelium could result in an ineffective host response and failure to eradicate potentially pathogenic organisms, leaving the host susceptible to colonisation, chronic inflammation and acute exacerbations.
This study examined the expression of TLR4 and HBD2 in respiratory epithelium in non-smokers and smokers with COPD. The effect of cigarette smoke was replicated in vitro by examining TLR4 mRNA and protein expression and quantifying IL-8 expression in airway epithelial cells stimulated with cigarette smoke extracts. The effects of other potential modulators of TLR expression in respiratory epithelium pertinent to COPD, including the long-acting beta2 agonist (LABA) salmeterol and the corticosteroids fluticasone and dexamethasone were also examined. The data indicate that altered expression of TLR4 may be important in the pathogenesis of COPD and may be modulated by corticosteroids, LABAs and cigarette smoke.
Materials and methods
Study population
Outpatients attending for upper GI endoscopies were recruited for nasal brush sampling following approval of study protocol and consent forms by the Beaumont Hospital Ethics Committee. Subjects were excluded on the basis of pre-existing immunosuppression, pulmonary or nasal pathology, including current or recent (within 6 weeks) upper or lower respiratory tract infection and reported normal functional status.
Nasal and Tracheobronchial Epithelial cell sampling
Following informed consent, nasal brushing was performed under direct vision using a Cervibrush + (CellPath plc)using a modification of the technique of Bridges et al [
8]. Tracheobronchial epithelial cells were harvested as in the method of Kelsen et al [
9]. Samples were accepted for analysis if they contained at least 80% epithelial cells.
Cell lines and culture
Human airway epithelial cells (A549, European Collection of Cell Cultures, Porton Down, UK) were cultured at 37°C in 5% CO2 in Ham's F12 (Gibco-BRL), 10% FCS, 1% penicillin/streptomycin. Prior to agonist treatment, cells were washed with serum-free F12 and placed under serum-free conditions or in serum containing 1% FCS for LPS stimulations.
Preparation of Fluticasone, Salmeterol and Dexamethasone
Fluticasone propionate and salmeterol were obtained from Glaxo SmithKline, Glaxo Wellcome UK Ltd, Stanley Park West, Uxbridge, Middlesex UB11 1BT, and reconstituted Ham's F12/0.01% DMA and Ham's F12/0.01% Methanol respectively to stock concentrations of 10-6 M. Dexamethasone was purchased from Sigma-Aldrich, Tallaght, Dublin, Ireland and reconstituted in 10% Ethanol in PBS to a stock concentration of 1 mM, and serial dilutions prepared in PBS.
Preparation of cigarette smoke extracts
Cigarette smoke extract (CSE) was freshly prepared for each experiment by a modification of a previously published method [
10]. Briefly, 2 filtered Marlboro Red cigarettes, each containing 0.8 mg of nicotine and 10 mg of tar according to the manufacturer's report, were bubbled through 20 ml serum free F-12 medium, pre-warmed to 37°C, by a mechanical vacuum pump. The extract was filtered through a 0.45 μm pore filter (Millipore, Bedford, MA) to remove bacteria and particles, and serial dilutions 1:10 were made.
Reverse Transcription (RT)-PCR
RNA isolation, cDNA synthesis and RTPCR were performed as previously described [
3] using gene-specific primers (Table
1). Products were analyzed by densitometry and compared in a semi quantitative manner relative to GAPDH using ImageMaster
® TotalLab Software (Amersham Pharmacia, Amersham, UK).
TLR4 (NM_003266) | | | |
F | AGATGGGGCATATCAGAGC | 569-587 | 481 bpa
|
R | GTCCATCGTTTGGTTCTGG | 1068-1050 | |
TLR4 (NM_003266)* | | | |
F | GGTGGAGCTGTACCGCCTT | 2982-3002 | 65 bp |
R | GCCCCAGGACACTGTCCTCCTC | 2697-2716 | |
TLR2 (U 88540) | | | |
F | TGCCCTGCCTATATGCAA | 381-398 | 486 bp |
R | GAACACATCGCTGACAACT | 936-918 | |
HBD2 (NM_AF071216) | | | |
F | GGTATAGGCGATCCTGTTACC TGC | 2688-2709 | 202 bp |
R | TCATGGCTTTTTGCAGCA TTTTGTTC | 4542-4567 | |
GAPDH (BC004109) | | | |
F | AACTCTGGTAAAGTGGAT | 122-138 | 211 bp |
R | TACTCAGCGCCAGCATCG | 333-316 | |
Real Time PCR
TLR4 mRNA was quantified using commercially available SYBR Green assays as previously described [
11] with primers listed in Table
1. The results are expressed as the ratio of the mean of triplicate target gene cDNA measurements to the triplicate housekeeping gene (β-actin) measurement.
Protein determination
IL-8 protein concentrations in cell supernatants were determined by sandwich ELISA (R & D Systems, U.K.). TLR4 protein was analysed in membrane and cytosolic fractions by Western Blot as previously described [
12] and surface expression by Laser Scanning Cytometry as previously described [
3].
Cell viability
Viability of A549 cells under stated treatment conditions was quantified using the Promega CellTiter 96 Aqueous One Solution Cell Proliferation Assay as recommended by the manufacturer.
Statistical analysis
Data were analyzed with GraphPad Prism 3.0 software package (GraphPad Software, San Diego, CA). Results are expressed as mean ± S.E. and were compared by Mann-Whitney test. Differences were considered significant when the P value was ≤ 0.05.
Discussion
Expression of TLR4 on respiratory epithelium allows rapid activation of host defense by pathogens, resulting in induction of inflammatory mediators and anti-microbial peptides, including HBD2. Recent evidence also implicates TLR4 deficiency in oxidant induced lung damage and emphysema [
17]. Here we report altered expression of TLR4 in the respiratory epithelium of smokers and in patients with COPD, and modifications associated with corticosteroid and LABA treatment that may contribute to our understanding of their therapeutic mechanisms.
Cigarette smoking is a major environmental risk factor predisposing to COPD and is also an independent risk factor for bacterial colonisation of the lower respiratory tract [
18,
19], acute respiratory infection [
20], and infective exacerbations of COPD [
21]. Our data demonstrates that smoking is associated with reduced TLR4 expression and LPS responsiveness in respiratory epithelium and is consistent with other data demonstrating reduced HBD2 production in response to LPS in respiratory epithelial cells following exposure to cigarette smoke [
22].
TLR4 and HBD2 expression was increased in subjects with mild-moderate COPD compared to normal controls, while with increasing severity of disease and fall in FEV1, expression was reduced. In contrast to alveolar macrophages [
23], TLR2 expression is not changed, suggesting that this is not a non-specific response to airway inflammation. There is little existing data regarding the transcriptional regulation of TLRs in human airway epithelial cells, although IFN-γ and TNFα have been shown to modulate TLR4 expression and function in human intestinal epithelium [
24,
25]. The inflammatory milieu in the airways in COPD includes many potential modulators of TLR4 including cytokines, acute phase reactants [
26,
27], proteases [
28], and anti-proteases [
29,
30] which may upregulate TLR4 in mild to moderate disease. Whether the reduced expression of TLR4 expression in severe COPD is an adaptive response to increased exposure to Gram-negative pathogens, as part of the phenomenon of endotoxin tolerance [
31] in an attempt to attenuate ongoing LPS induced airway inflammation, or pre-exists and thus promotes colonisation [
32] is not clear. Reduced epithelial expression of TLR4 may represent a useful biomarker of disease severity.
Our COPD population differed from controls in terms of their exposure to inhaled medications, namely LABAs and corticosteroids. We therefore went on to explore the potential of these compounds to modulate TLR4 expression
in vitro. Glucocorticoids have been previously reported to modulate lung responses to infection, including Pseudomonas [
33]. There have been no previous reports about the effect of corticosteroids on TLR4 expression in epithelial cells. Here we demonstrate that corticosteroid exposure, at clinically relevant doses [
34,
35] results in downregulation of TLR4 and impaired IL-8 response to LPS. Here we provide evidence for a mechanism whereby corticosteroids could impair host defence against Gram-negative bacteria by downregulation of TLR4 expression.
LABAs such as salmeterol are prescribed primarily as bronchodilators, although accumulating evidence in recent years indicates that LABAs have numerous anti-inflammatory properties [
36]. Beta-2 adrenergic receptors are expressed in respiratory epithelium, but the immunomodulatory effect of LABAs on these cells has been largely unexplored. Here we show that the LABA salmeterol had no effect on TLR4 gene transcription or total protein expression, but did induce membrane presentation of TLR4 from the cytoplasmic/nuclear compartment. A similar post-translational effect has been described in nasal epithelium of patients with allergic rhinitis compared to healthy subjects [
37], while nuclear localisation of TLR4 has been confirmed in bronchial epithelium [
38]. TLR4 has been shown to cycle rapidly between the Golgi and the membrane, with signal transduction occurring only at the membrane [
39]. Little is known about the mechanism of this translocation or indeed transport from the nucleus. Our data, demonstrating a beta-receptor mediated effect on post-translational TLR4 transport suggests a potential role for cAMP-dependent protein kinases in this process.
Following in vivo inhalation of 50 μ of salmeterol, the estimated lung tissue concentrations are between 10
-7 and 10
-8 M [
40], and local concentrations at the site of deposition of the drug namely the epithelium are likely higher. The observed effects at doses of 10
-7 and 10
-6 are therefore clinically relevant.
While the anti-inflammatory effects of corticosteroids are well documented, chronic inhaled corticosteroid therapy alone has failed to impact significantly on disease progression or mortality in numerous large scale multi-centre placebo controlled trials of inhaled corticosteroids in COPD [
41‐
45]. Downregulation of TLR4 membrane protein expression and consequent susceptibility to Gram-negative infection may contribute to the failure of unopposed steroid therapy in these trials. Abrogation of this effect by the addition of salmeterol may represent another important advantage of co-prescription of these compounds, and may contribute to the clinically important improvements in outcome which result when these compounds are prescribed together. In the recent TORCH study, combination therapy with fluticasone and salmeterol resulted in significant reductions in exacerbation rate and 3-year mortality (both COPD related and all cause) compared to fluticasone alone, which had no effect on mortality compared to placebo [
46].
In the presence of CSE, the protective effect of salmeterol on TLR4 signalling is lost and in fact there is a small but statistically significant further reduction in LPS-induced IL-8 expression compared to dexamethasone alone. These findings are in keeping with recent report that combination of fluticasone and salmeterol potentiates the suppression of cigarette smoke-induced IL-8 production by macrophages [
15]. Although salmeterol was found to have no effect on CSE induced IL-8 production in airway smooth muscle cells [
16], although the effect of LPS was not examined in these studies. It would be of great interest to know if the clinical effects of salmeterol and fluticasone in combination were more profound in smokers compared to non-smokers in the TORCH study [
46], but this subgroup analysis has not been reported.
The respiratory epithelium is in constant dynamic interaction with the environment, and is uniquely exposed to airborne pathogens and toxins, as well as aerosolised drugs. The TLRs perform a pivotal role in host defence, and this study demonstrates that TLR4 expression in respiratory epithelium is altered in COPD, potentially contributing to the airway inflammation and infective exacerbations which characterise this disease. TLR4 expression is modulated both by drugs used to treat airways inflammation and by cigarette smoke, the major pathogenic determinant of COPD. A greater understanding of the mechanism of these effects may improve our understanding of the pathogenesis of airways disease, and direct future therapies.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RMacR carried out patient recruitment, sample collection and analysis, gene and protein expression analysis, drafted the manuscript and contributed to study design and analysis. CG carried out LSC, cell viability studies and immunoassays, and contributed to manuscript preparation, study design and analysis. DD, NmcE and SON contributed to study design, analysis and manuscript preparation. All authors read and approved the final manuscript.