Abstract

The numbers of macrophages are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. COPD lung macrophages have reduced ability to phagocytose microbes and efferocytose apoptotic cells. Inhaled corticosteroids (ICSs) are widely used anti-inflammatory drugs in COPD; however, their role beyond suppression of cytokine release has not been explored in COPD macrophages. We have examined the effects of corticosteroids on COPD lung macrophage phenotype and function.

Lung macrophages from controls and COPD patients were treated with corticosteroids; effects on gene and protein expression of CD163, CD164, CD206, MERTK, CD64, CD80 and CD86 were studied. We also examined the effect of corticosteroids on the function of CD163, MERTK and cluster of differentiation 64 (CD64).

Corticosteroid increased CD163, CD164, CD206 and MERTK expression and reduced CD64, CD80 and CD86 expression. We also observed an increase in the uptake of the haemoglobin–haptoglobin complex (CD163) from 59 up to 81% and an increase in efferocytosis of apoptotic neutrophils (MERTK) from 15 up to 28% following corticosteroid treatment. We observed no effect on bacterial phagocytosis.

Corticosteroids alter the phenotype and function of COPD lung macrophages. Our findings suggest mechanisms by which corticosteroids exert therapeutic benefit in COPD, reducing iron available for bacterial growth and enhancing efferocytosis.

Introduction

Lung macrophages sense and remove microbes and apoptotic cellular debris by phagocytosis and efferocytosis, respectively [1,2]. Lung macrophages also mount both pro- and anti-inflammatory cytokine responses to external triggers including microbes and oxidative stress [3,4]. In healthy individuals, these macrophage functions enable host defence against microbes and maintenance of lung homoeostatic balance [5].

Chronic obstructive pulmonary disease (COPD) is characterised by a complex and persistent inflammatory response to the inhalation of noxious particles, commonly from cigarette smoking [6]. Lung macrophage numbers are increased in COPD [7–9], contributing to the inflammatory burden through the secretion of cytokines and chemokines in response to pathogens, particulate matter and oxidative stress signals. COPD macrophages have reduced phagocytosis and efferocytosis ability [10,11]. The homoeostatic function of COPD macrophages is therefore altered, skewing towards a phenotype causing inflammation and with reduced ability to clear microbes and apoptotic material.

Inhaled corticosteroids (ICSs) are commonly used anti-inflammatory drugs in COPD. Corticosteroids reduce pro-inflammatory mediator secretion from COPD alveolar macrophages exposed to microbial or oxidative stress triggers [12,13]. However, the effects of corticosteroids on other aspects of COPD macrophage function remain less well-understood.

Macrophages display phenotypic cellular markers that indicate different functionality [14,15]. These include cluster of differentiation 64 (CD64, also known as Fc-γ receptor 1) which is involved in the recognition and uptake of opsonised bacteria [16], CD163 which recognises and mediates the uptake of haemoglobin–haptolgobin complexes [17], MERTK which mediates apoptotic cell uptake [18] and CD164 which is involved in cell adhesion and migration [19]. Macrophage phenotype is altered in COPD patients compared with controls [20–22]. For example, the expression level of CD206 (involved in bacterial recognition) on COPD macrophages is lower compared with controls [20,23]. Furthermore, current cigarette smoking suppresses the expression of CD14, CD163 and CD206 on COPD macrophages [20].

Studies in monocyte-derived macrophages (MDMs) from healthy donors have shown that corticosteroids increase the expression of CD163 and CD163-mediated uptake of haemoglobin–haptoglobin complexes [17,24]. In addition, corticosteroid treatment of COPD MDMs prevented bacterial induced reductions in various cell surface receptors, while having no effect on bacterial phagocytosis [25,26]. However, COPD lung macrophages are functionally unique cells, with phenotype and function being influenced by their microenvironment [14,20]. While MDMs are useful macrophage models, they do not fully display the heterogeneity and functional characteristics of lung macrophages [10].

There is a paucity of information regarding the effects of corticosteroids on COPD lung macrophage phenotype and function beyond inhibition of cytokine production. We have previously reported gene expression data of COPD MDMs treated with corticosteroids, showing reductions in CD64, CD80 and CD86 expression and increases in CD163, CD164, CD206 and MERTK expression [27]. Given the limited similarity between COPD MDMs and lung macrophages, we wanted to now investigate these changes using COPD lung macrophages.

The aim of the current study was to examine the effects of corticosteroids on the phenotype and function of COPD lung macrophages. The present study provides information beyond the previously described anti-inflammatory effects on cytokine production [12,13]. We used real-time qPCR and flow cytometry to analyse gene and protein expression changes, respectively, following corticosteroid treatment. We used a hypothesis-driven approach using pre-selected targets based upon gene expression profiling data from our previous study [27]. We also assessed the functional effect of changes to some of these targets by examining haptolgobin-haemoglobin complex uptake (CD163), efferocytosis of apoptotic neutrophils (MERTK) and opsonic bacterial phagocytosis (CD64) following corticosteroid treatment.

Methods

Study subjects

Sixty-nine patients undergoing surgical resection for suspected lung cancer were recruited. COPD was diagnosed based on GOLD recommendations [6]. Controls were smokers without airflow limitation. Ex-smokers were defined as individuals who had stopped smoking for ≥1 year. This research was approved by NRES Committee North West-Greater Manchester South (reference 03/SM/396) and all experiments were performed in accordance with relevant guidelines and regulations. This research has been carried out in accordance with the World Medical Association Declaration of Helsinki and all subjects provided written information consent.

Lung macrophage isolation

Lung macrophages were isolated from the lungs as previously described [28]. Following isolation, macrophages were left to adhere for 16 h prior to removal of non-adherent cells the following day. The order of experiments were as follows: gene expression studies, protein expression study, functional studies (CD163, MERTK, CD64 respectively). Not all studies have the same number of samples due to sample availability.

Corticosteroid effects on lung macrophage gene expression

Macrophages were treated with dexamethasone, budesonide, fluticasone propionate (1–100 nM) or DMSO control (all Sigma–Aldrich) for 6 h. Culture supernatants were removed and cells were lysed in RLT buffer. Total RNA was purified from cell lysates using RNeasy kits (Qiagen, Crawley, U.K.) according to manufacturer’s instructions. DNA contamination was prevented by on-column addition of DNase (Qiagen, Crawley, U.K.) according to manufacturer’s instructions. Reverse transcription was performed on 50 ng of RNA using the Verso cDNA kit (Thermo Scientific). The resulting cDNA was reacted with ABsolute blue qPCR mix (Thermo Scientific) in 25 µl reactions containing premade ABI Taqman gene expression assays for CD163 (Hs00174705_m1), CD164 (Hs00174789_m1), CD206 (Hs00267207_m1), MERTK (Hs01031973_m1), CD64 (Hs02340031_m), CD80 (Hs01045163_m1), CD86 (Hs01567026_m1) and the endogenous control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalogue no.: 4352934E) (Applied Biosystems, Warrington, U.K.). Controls without RT-enzyme showed there was no genomic DNA amplification. Thermal cycling was carried out on a Stratagene MX3005P (Agilent Technologies, West Lothian, U.K.). Relative expression levels were determined using the 2−ΔΔCt (untreated vs corticosteroid treated) or 2−ΔCt (baseline expression).

Corticosteroid effects on CD163, MERTK and CD64 protein expression

Macrophages were treated with dexamethasone (100 nM) for 24 h, followed by a phosphate-buffered saline (PBS) wash and manual dissociation from the culture plate. Macrophages were washed in PBS/2% bovine serum albumin (BSA), re-suspended and incubated for 30 min with either 5 µl CD64 APC-Fire™750 (BioLegend, clone: 10.1), 5 µl CD163 PE (Thermo Fisher, clone: eBioGHI/61 (GHI/61) or MERTK APC-Fire™750 (BioLegend, clone: 590H11G1E3). Macrophages were washed then re-suspended in PBS/2% BSA and acquired on FACSCanto II.

Culture, opsonisation and phagocytosis of bacteria

Non-typeable Haemophilus influenzae (NTHi; strain NCTC 12699) and Moraxella catarrhalis (M. catarrhalis; strain NCTC 11020, supplied by Public Health England) were grown on chocolate agar, supplemented with 5% horse defibrinated blood (E and O Laboratories Ltd), overnight at 37°C and 5% CO2 in air. The growth from each plate was re-suspended in 2.5 ml brain heart infusion broth (BHI) for M. catarrhalis and BHI supplemented with hemin (10 µg/ml) and nicotinamide adenine dinucleotide (10 µg/ml) (sBHI) for NTHi. The optical density at 600 nm (OD600 nm) was determined for the 2.5 ml culture, which was then used to calculate the volume of inoculum required to give a starting OD600 nm of 0.01 in BHI and sBHI. Cultures were incubated at 37°C and 5% CO2 in air until an OD600 nm of 0.4–0.7 was achieved (mid-log phase). Bacteria were then re-suspended to 1.2 at OD600 nm. Bacteria were washed once in PBS before re-suspending the pellets in 1 ml PBS. The bacterial concentration was calculated assuming that an OD600 of 1.2 corresponds to 108 colony forming units. Bacteria were heat killed at 90°C for 30 min prior to labelling with 100 nM with BacLight Red (Thermo Fisher) according to manufacturer’s instructions.

Peripheral blood was collected in an SST vacutainer (BD Biosciences) from five healthy never-smokers and processed according to manufacturer’s instructions. The serum was collected and heat-inactivated at 56°C for 30 min, pooled and stored at −80°C. Bacteria were opsonised using 10% heat-inactivated human serum for 30 min at room temperature. Opsonisation was confirmed by positive IgG staining analysed by flow cytometry. Bacteria were then washed in 2% BSA in PBS and re-suspended in RPMI prior to use.

Macrophages were treated with 100 nM dexamethasone or RPMI for 24 h. The following day, medium was removed and BacLight Red (Thermo Fisher) labelled NTHi or M. catarrhalis were added at a ratio of 100:1, or pHrodo red Staphylococcus aureus bioparticles (Invitrogen) were added at a concentration of 0.05 mg/ml. Cells were incubated with bacteria for 90 min at 37°C and 5% CO2 in air. Macrophages were removed from the plate by manual dissociation and re-suspended in 2% BSA in PBS prior to incubation with CD64 APC-Fire™750 (BioLegend, clone: 10.1) for 30 min at room temperature. Cells were then washed, re-suspended in 2% BSA in PBS and acquired on FACSCanto II.

Haptoglobin–haemoglobin complex uptake assay

Haemoglobin (Sigma) was fluorescently labelled with the Alexa Fluor 633 protein labelling kit (Thermo Fisher) according to manufacturer’s instructions. Labelled haemoglobin was incubated with equal amounts of haptoglobin (Sigma) at 37°C for 30 min to form the haptoglobin–haemoglobin complex.

Macrophages were treated with 100 nM dexamethasone or RPMI for 24 h. The following day, medium was removed and the haptoglobin–haemoglobin complex (50 µg/ml) was added. Cells were incubated with the complex for 4 h at 37°C and 5% CO2 in air. Macrophages were removed from the plate by manual dissociation and re-suspended in 2% BSA in PBS prior to incubation with CD163 PE (clone: eBioGHI/61 (GHI/61) for 30 min at room temperature. Cells were then washed, re-suspended in 2% BSA in PBS and acquired on FACSCanto II.

Efferocytosis of apoptotic neutrophils

Neutrophils were isolated from the peripheral blood by dextran sedimentation as previously described [29]. A single donor was used and blood was taken the day before lung macrophage samples were available. Isolated neutrophils were labelled with Mitotracker red (Thermo Fisher) according to manufacturer’s instructions, washed and re-suspended in RPMI-1640 media supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin overnight at 37°C and 5% CO2 in air to induce apoptosis. The following day, annexin and propidium iodide expression were analysed by flow cytometry.

Macrophages were treated with 100 nM dexamethasone or RPMI for 24 h. The following day, medium was removed and apoptotic neutrophils were added at a ratio of 1:1 for 60 min at 37°C and 5% CO2 in air. Neutrophils not taken up were removed and macrophages were removed from the plate by manual dissociation and re-suspended in 2% BSA in PBS prior to incubation with MERTK APC-Fire™ 750 (clone: 590H11G1E3) for 30 min at room temperature. Cells were then washed, re-suspended in 2% BSA in PBS and acquired on FACSCanto II.

Statistical analysis

All statistical analyses were performed using GraphPad InStat software (GraphPad Software Inc., La Jolla, California, U.S.A.). Data distributions were determined by the D’Agostino and Pearson normality test. Parametric data were compared using a one-way ANOVA followed by Tukey’s post-hoc analysis or an unpaired t test where only two groups were compared. Non-parametric data were compared using a Kruskal–Wallis test followed by a Dunn’s post-hoc analysis or a Mann–Whitney test where only two groups were compared. A two-way ANOVA was used to analyse comparisons between steroids.

Results

Demography

The study population characteristics are shown in Table 1. COPD patients had significantly lower forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) ratio compared with controls. The COPD group had a higher number of current smokers but the pack-year histories were similar between groups.

Table 1
Demographics of the study population
ControlsCOPDP value
n 33 36 N/A 
Age (years) 68 (8) 68 (6) >0.05 
Gender: male (%) 58 35 >0.05 
FEV1 (L) 2.6 (0.6) 1.7 (0.5) <0.001 
FEV1% predicted 101 (16) 77 (19) <0.001 
FVC (l) 3.5 (0.9) 3.0 (0.7) 0.02 
FEV1/FVC ratio (%) 79 (13) 59 (9) <0.001 
Current smokers (%) 30 58 0.01 
Pack-year history 39 (23) 48 (33) 0.3 
ICS users (%) N/A 33 N/A 
LABA users (%) N/A 14 N/A 
LAMA users (%) N/A 31 N/A 
ControlsCOPDP value
n 33 36 N/A 
Age (years) 68 (8) 68 (6) >0.05 
Gender: male (%) 58 35 >0.05 
FEV1 (L) 2.6 (0.6) 1.7 (0.5) <0.001 
FEV1% predicted 101 (16) 77 (19) <0.001 
FVC (l) 3.5 (0.9) 3.0 (0.7) 0.02 
FEV1/FVC ratio (%) 79 (13) 59 (9) <0.001 
Current smokers (%) 30 58 0.01 
Pack-year history 39 (23) 48 (33) 0.3 
ICS users (%) N/A 33 N/A 
LABA users (%) N/A 14 N/A 
LAMA users (%) N/A 31 N/A 

Abbreviations: LABA, long-acting β-2 agonist; LAMA, long-acting muscarinic antagonist. Data are presented as mean (SD).

Corticosteroid effects on macrophage gene expression

In lung macrophages from 14 controls and 19 COPD patients, dexamethasone (100 nM) significantly increased the expression of CD163, CD206, MERTK and CD164 and significantly reduced expression of CD64, CD80 and CD86 compared with untreated cells (Figure 1). Gene expression levels in dexamethasone treated or untreated cells were similar in COPD patients compared with controls (Supplementary Figure S1).

Corticosteroid effects on lung macrophage gene expression

Figure 1
Corticosteroid effects on lung macrophage gene expression

Macrophages from 14 controls (white bars) and 19 COPD patients (black bars) were treated with 100 nM dexamethasone for 6 h and the gene expression of CD163, CD164, CD206 and MERTK (A), CD64, CD80 and CD86 (B) were assessed by RT-qPCR. Comparisons were made with untreated macrophages (red dotted line) where *** = significant difference (P<0.001). Data presented as mean + SEM.

Figure 1
Corticosteroid effects on lung macrophage gene expression

Macrophages from 14 controls (white bars) and 19 COPD patients (black bars) were treated with 100 nM dexamethasone for 6 h and the gene expression of CD163, CD164, CD206 and MERTK (A), CD64, CD80 and CD86 (B) were assessed by RT-qPCR. Comparisons were made with untreated macrophages (red dotted line) where *** = significant difference (P<0.001). Data presented as mean + SEM.

A subanalysis of COPD ICS users and non-users showed similar gene expression changes in the two groups (Supplementary Figure S2), apart from CD164 which had significantly increased expression in the ICS users compared with non-users following dexamethasone treatment (P<0.05). In untreated macrophages (Supplementary Figure S3), there were no significant differences between groups.

We compared the effect of dexamethasone, budesonide and fluticasone propionate (1–100 nM) on lung macrophage gene expression from ten COPD patients (Figure 2). A similar pattern of results was observed for these corticosteroids, with a trend for dexamethasone causing smaller changes at some concentrations and budesonide causing greater changes at some concentrations, although these differences were small.

Comparison of corticosteroid effects on COPD lung macrophage gene expression

Figure 2
Comparison of corticosteroid effects on COPD lung macrophage gene expression

Macrophages from ten COPD patients were treated with dexamethasone (red), budesonide (blue) or fluticasone propionate (green) (all 1–100 nM) for 6 h and the gene expression of CD163 (A) CD164 (B) CD206 (C) MERTK (D) CD64 (E) CD80 (F) and CD86 (G) was assessed by RT-qPCR. Comparisons were made between corresponding concentrations where * is dexamethasone vs budesonide, ¶ is dexamethasone vs fluticasone propionate and † is budesonide vs fluticasone propionate. One, two and three symbols correspond to P<0.05, P<0.01 and P<0.001, respectively. Data presented as mean ± SEM.

Figure 2
Comparison of corticosteroid effects on COPD lung macrophage gene expression

Macrophages from ten COPD patients were treated with dexamethasone (red), budesonide (blue) or fluticasone propionate (green) (all 1–100 nM) for 6 h and the gene expression of CD163 (A) CD164 (B) CD206 (C) MERTK (D) CD64 (E) CD80 (F) and CD86 (G) was assessed by RT-qPCR. Comparisons were made between corresponding concentrations where * is dexamethasone vs budesonide, ¶ is dexamethasone vs fluticasone propionate and † is budesonide vs fluticasone propionate. One, two and three symbols correspond to P<0.05, P<0.01 and P<0.001, respectively. Data presented as mean ± SEM.

Smoking status did not affect the outcome of the results for Figures 1 and 2 (data not shown).

Corticosteroid effects on CD163, MERTK and CD64 protein levels

We selected three candidate genes for further characterisation at the protein level: CD163, MERTK and CD64. These were selected based on higher fold changes following corticosteroid treatment and the suitability for functional studies. Lung macrophages from nine controls and seven COPD patients were treated with dexamethasone for 24 h and CD163, MERTK and CD64 protein expression measured by flow cytometry (Figure 3). The mean percentage of CD163+ macrophages increased from 9 to 27% and from 13 to 27% following dexamethasone treatment in the controls and COPD patients, respectively (P<0.05). There was a numerical trend (not statistically significant) for increased MFI of CD163+ macrophages.

Corticosteroid effects on CD163, MERTK and CD64 protein expression

Figure 3
Corticosteroid effects on CD163, MERTK and CD64 protein expression

Macrophages from nine controls (white bars) and seven COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h (A–D,G,H) or 48 h (E,F) and CD163 (A,B), MERTK (C–F) and CD64 (G,H) expression were assessed by flow cytometry. The percentage of positive macrophages (A,C,E,G) and median fluorescence intensity (B,D,F,H) are reported. Comparisons were made between untreated and dexamethasone treated cells where * = significant difference (P<0.05). Data presented as mean + SEM.

Figure 3
Corticosteroid effects on CD163, MERTK and CD64 protein expression

Macrophages from nine controls (white bars) and seven COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h (A–D,G,H) or 48 h (E,F) and CD163 (A,B), MERTK (C–F) and CD64 (G,H) expression were assessed by flow cytometry. The percentage of positive macrophages (A,C,E,G) and median fluorescence intensity (B,D,F,H) are reported. Comparisons were made between untreated and dexamethasone treated cells where * = significant difference (P<0.05). Data presented as mean + SEM.

There were no differences in the mean percentage or MFI of MERTK+ macrophages following dexamethasone treatment for 24 h in both patient groups. However, when treated for 48 h there was a significant increase in the mean percentage of MERTK+ macrophages from 54 to 63% in the control group (P<0.05; Figure 3). There were no differences in the MFI between treated and untreated cells.

The percentage of CD64+ macrophages significantly reduced from 49 to 46% following dexamethasone treatment in the COPD group only (P<0.05). There was no change in the MFI of CD64+ macrophages in either group.

There was no difference in the expression of CD163, MERTK or CD64 between control and COPD macrophages. Smoking status did not affect the outcome of the results (data not shown).

Corticosteroid effects on the uptake of the haptoglobin–haemoglobin complex

To examine the impact of increased CD163 expression following dexamethasone treatment, we measured the uptake of the haptoglobin–haemoglobin complex. In lung macrophages from five controls and five COPD patients, the uptake of the haptoglobin–haemoglobin complex was significantly increased following dexamethasone treatment compared with untreated cells (Figure 4). Uptake increased from 43 to 69% in control macrophages and 59 to 81% in COPD macrophages. In addition, the mean percentage of CD163+ macrophages significantly increased from 36 to 70% in controls and 55 to 72% in COPD patients (Figure 4). There were no statistically significant differences between the two groups.

Corticosteroid effects on the uptake of the haptoglobin–haemoglobin complex

Figure 4
Corticosteroid effects on the uptake of the haptoglobin–haemoglobin complex

Macrophages from five controls (white bars) and five COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to the haptoglobin-haemoglobin complex for 60 min. The percentage of haptoglobin-haemoglobin complex+ macrophages (A) and the percentage of CD163+ macrophages (B) are reported. Comparisons were made between untreated and dexamethasone treated cells were *, ** and *** = significant difference (P<0.05, P<0.01 and P<0.001 respectively). Data are presented as mean + SEM.

Figure 4
Corticosteroid effects on the uptake of the haptoglobin–haemoglobin complex

Macrophages from five controls (white bars) and five COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to the haptoglobin-haemoglobin complex for 60 min. The percentage of haptoglobin-haemoglobin complex+ macrophages (A) and the percentage of CD163+ macrophages (B) are reported. Comparisons were made between untreated and dexamethasone treated cells were *, ** and *** = significant difference (P<0.05, P<0.01 and P<0.001 respectively). Data are presented as mean + SEM.

Corticosteroid effects on the efferocytosis of apoptotic neutrophils

To investigate the impact of increased MERTK expression following dexamethasone treatment, we measured the efferocytosis of apoptotic neutrophils. Neutrophil apoptosis was induced by overnight culture. The proportion of neutrophils undergoing early apoptosis (annexin+ PI) and late apoptosis (annexin+ PI+) was 73 and 13%, respectively (data not shown).

Dexamethasone treatment significantly increased the uptake of apoptotic neutrophils by control (n=5) and COPD (n=5) lung macrophages (Figure 5). Uptake increased from 27 to 32% in controls and 15 to 28% in COPD patients. There was a significant increase in the number of MERTK+ macrophages from 7 to 16% in the COPD group only (Figure 5). There were no significant differences between controls and COPD patients.

Corticosteroid effects on the efferocytosis of apoptotic neutrophils

Figure 5
Corticosteroid effects on the efferocytosis of apoptotic neutrophils

Macrophages from five controls (white bars) and five COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to Mitotracker labelled apoptotic neutrophils for 60 min. The percentage of macrophages containing Mitotracker labelled neutrophils (A) and the percentage of MERTK+ macrophages (B) are reported. Comparisons were made between untreated and dexamethasone treated cells where * and ** = significant difference (P<0.05 and P<0.01, respectively). Data presented as mean + SEM.

Figure 5
Corticosteroid effects on the efferocytosis of apoptotic neutrophils

Macrophages from five controls (white bars) and five COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to Mitotracker labelled apoptotic neutrophils for 60 min. The percentage of macrophages containing Mitotracker labelled neutrophils (A) and the percentage of MERTK+ macrophages (B) are reported. Comparisons were made between untreated and dexamethasone treated cells where * and ** = significant difference (P<0.05 and P<0.01, respectively). Data presented as mean + SEM.

Corticosteroid effects on the phagocytosis of opsonised bacteria

Due to reductions in CD64 gene expression and protein levels following dexamethasone treatment, we examined the effect of corticosteroid treatment on the phagocytosis of opsonised bacteria. Dexamethasone treatment did not alter the phagocytosis of opsonised NTHi or M. catarrhalis in both controls (n=8) and COPD patients (n=4) (Figure 6). The levels of CD64 did not significantly change in these experiments (Figure 6).

Corticosteroid effects on the phagocytosis of opsonised bacteria

Figure 6
Corticosteroid effects on the phagocytosis of opsonised bacteria

Macrophages from eight controls (white bars) and four COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to BacLight labelled, opsonised NTHi (A,B) or M.catarrhalis (C,D) for 90 min. The percentage of of BacLight+ macrophages (A,C) and the percentage of CD64+ macrophages (B,D) are reported. Data are presented as mean + SEM.

Figure 6
Corticosteroid effects on the phagocytosis of opsonised bacteria

Macrophages from eight controls (white bars) and four COPD patients (black bars) were treated with 100 nM dexamethasone for 24 h prior to exposure to BacLight labelled, opsonised NTHi (A,B) or M.catarrhalis (C,D) for 90 min. The percentage of of BacLight+ macrophages (A,C) and the percentage of CD64+ macrophages (B,D) are reported. Data are presented as mean + SEM.

The baseline level of phagocytosis for M. catarrhalis was numerically lower in COPD patients compared with controls but this did not reach statistical significance. Phagocytosis of NTHi was similar when comparing groups.

These findings were corroborated using a different system to analyse bacterial phagocytosis; dexamethasone treatment did not impact the phagocytosis of opsonised pHrodo-labelled S. aureus bioparticles in both control and COPD macrophages (Supplementary Figure S4). The baseline level of phagocytosis was numerically lower in COPD patients compared with controls but this did not reach significance.

Discussion

Corticosteroids have a wide range of actions. We used COPD lung macrophages to investigate corticosteroid effects beyond the modulation of cytokine secretion. Corticosteroids reduced CD64, CD80 and CD86 gene expression and increased CD163, CD164, CD206 and MERTK gene expression in COPD lung macrophages. We then selected three of these genes for protein and functional studies, based on magnitude of fold change and practicality of performing functional studies. CD163 showed a clear up-regulation of both protein expression and function (uptake of haptoglobin-haemoglobin), while MERTK showed a small but inconsistent increase in protein expression, which nevertheless was associated with increased efferocytosis of apoptotic neutrophils. In contrast, there was a small and inconsistent decrease in CD64 protein levels which was not accompanied with any change in bacterial phagocytosis. These functional studies have potential clinical relevance, demonstrating that corticosteroids influence COPD macrophage ability to regulate iron bioavailability and efferocytosis, while not modulating phagocytosis.

Macrophage plasticity is commonly described by the M1/M2 polarisation paradigm, with further subdivision into M2a, M2b, M2c and M2d subsets [30]. However, macrophages can express markers that fit into both M1-like and M2-like phenotypes [31]. This suggests the M1/M2 paradigm is too simplistic and that macrophages form heterogeneous subpopulations with different physiological functions influenced by location and the microenvironment [20]. Nevertheless, corticosteroids have been shown to induce an M2c subset of macrophages characterised by increased expression of CD163, CD206 and MERTK [32,33]. Such studies have used MDMs from healthy patients [34,35]. Our results support these previous observations, but now using COPD lung macrophages; corticosteroid treatment reduced CD80 and CD86 gene expression, which are associated with M1-like macrophages, and increased CD163, CD206 and MERTK expression, which are associated with M2c macrophages. This subset of macrophages is associated with immune suppression and tissue repair [32]. Our results indicate that ICS use causes COPD macrophages to skew towards a phenotype with an increased focus on the restriction of tissue damage and/or tissue repair.

Bacteria use iron available from the host for growth and survival [36]. Indeed, iron levels are increased in the lungs of COPD patients [37]. The corticosteroid induced increase in haptoglobin-haemoglobin uptake, associated with increased CD163 expression, suggests a mechanism to reduce iron bioavailability and thereby restrict bacterial growth. On the other hand, it is known that macrophage phagocytosis is reduced in COPD [10]. However, corticosteroids did not alter macrophage phagocytosis, despite some evidence for a reduction in CD64 expression levels. Overall, we demonstrated a corticosteroid-dependent mechanism whereby macrophages could restrict bacterial growth, and that corticosteroids can enhance efferocytosis. These results demonstrate potentially beneficial effects of corticosteroids on COPD lung macrophages beyond the well documented anti-inflammatory effects on cytokine secretion [12,13].

Macrophages employ several mechanisms to clear extracellular iron, including uptake of transferrin- and lactoferrin-bound iron and uptake of haemoglobin via the haptoglobin-CD163 route [38]. Under homoeostatic conditions the clearance of extracellular iron is handled mainly by transferrin and the role of haptoglobin-mediated uptake is likely to be low [39]. However during inflammation and infection when the bioavailability of iron increases, haptoglobin-mediated uptake increases [40]. Our results suggest that ICS use may increase haemoglobin uptake by lung macrophages and thereby reduce bioavailability of iron for bacteria. This may reduce bacterial colonisation. Iron is also a potent catalyst for the production of reactive oxygen species (ROS) via the Fenton reaction [41]. ROS are increased in the lungs of COPD patients and are capable of causing tissue destruction if produced in excess [42]. ICS may therefore reduce the impact of iron as a catalyst for ROS production and subsequent ROS mediated tissue damage in COPD patients.

The numbers of apoptotic cells are increased in the lungs of COPD patients [43]. These cells can cause tissue damage and inflammation by the release of cellular material. The uptake of apoptotic cells by COPD lung macrophages is lower compared with controls [11]. Our findings suggest that ICS may increase apoptotic cell uptake by increasing MERTK expression in lung macrophages. This may help reduce inflammation and further tissue damage.

ICS use is associated with increased incidence of pneumonia in COPD patients [44]. This may be due to the suppression of anti-viral immunity by ICS and increased susceptibility to secondary bacterial infections [45]. We report that corticosteroid treatment of lung macrophages does not reduce opsonic bacterial phagocytosis despite reducing gene expression levels of CD64. The two-fold reduction in CD64 gene expression following corticosteroid treatment was associated with a small effect on protein expression. Furthermore, macrophages are equipped with alternative pathways to phagocytose bacteria including CD206 which increased during our gene expression experiments. It has previously been reported that corticosteroids do not alter the phagocytosis of non-opsonised NTHi and Streptococcus pneumonia by COPD MDMs [25]. MDM phenotype and function do not always match those of lung macrophages [10], so we used COPD lung macrophages with opsonised bacteria, which is arguably more physiologically relevant because the lungs contain large amounts of immunoglobulins [46]. Collectively, these observations suggest ICS do not suppress bacterial phagocytosis. However, it is uncertain whether this affects pneumonia risk in COPD patients taking ICS; fluticasone propionate has previously been shown to down-reguate MHC class I and II receptors in MDMs [47]. This may interfere with antigen presentation and cause increased susceptibility to infection. We also show reduced CD80 and CD86 gene expression following corticosteroid treatment, important co-stimulatory molecules for lymphocyte activation.We isolated macrophages from lung tissue as far distal to the tumour as possible from individuals with lung cancer. This is a commonly used method in COPD research [12,48,49]. We have previously shown that the corticosteroid response of macrophages isolated from the bronchoalveolar lavage of patients without lung cancer did not differ from macrophages isolated from the lung tissue of cancer patients [12]. However, we cannot rule out the possibility that cancer influenced the phenotype of the macrophages that we collected. Before experimentation we cultured the macrophages overnight (approximately 16 h). We have previously shown that levels of phosphorylated p38 MAPK are high in freshly isolated macrophages, and overnight culture allows this to reduce over time [28]. This p38 MAPK activation likely arises through mechanical or osmotic cell stress that occurs during macrophage isolation, and it seems important to avoid such experimental artefacts by allowing macrophages to ‘rest’ before further experimentation.

Although dexamethasone is an ICS used for COPD treatment, it is widely used as a tool compound for cellular studies [50,51]. The pharmacological properties of budesonide and fluticasone propionate are different, including solubility and efficacy [52]. To avoid restricting the results to just one of these corticosteroids, we opted to use dexamethasone. We replicated our PCR data using different corticosteroids. Overall, the differences between the corticosteroids were small. This supports the use of dexamethasone as a tool compound for the functional experiments. We chose a concentration of 100 nM dexamethasone for the majority of our experiments as this is a physiologically relevant concentration; it was previously shown that the concentration of inhaled fluticasone propionate in lung tissue was up to 12 nmol/kg [53]. More recently developed inhalers deliver more drug to the lungs, and tissue concentrations may be lower than that achieved in the epithelial lining fluid. Nevertheless, the concentration we chose was in the clinically relevant range.

Although there were some numerical differences (CD206 and CD80), we did not observe any significant differences in gene expression between COPD patients using and not using ICS. The sensitivity of this analysis will be affected by the concentration of ICS present in the distal lung regions. This varies between individuals [53] and is influenced by several factors including drug formulation, inhaler technique and also compliance. Similarly, some patients were using long acting bronchodilators, which have anti-inflammatory actions in vitro [54]. However, there is no clinical data to demonstrate such effects in COPD patients, and the variable deposition of these drugs in the distal lung makes it unlikely that the use of these drugs had any major effect on our results.

In conclusion, we have shown that corticosteroids can alter the phenotype and function of lung macrophages. We report two important findings using COPD lung macrophages; increased uptake of haptoglobin-haemoglobin and increased efferocytosis of apoptotic neutrophils following corticosteroid treatment. We also add to previous observations, in MDMs, that corticosteroids do not alter bacterial phagocytosis. The modulation of iron bioavailability and the enhancement of efferocytosis may contribute to the therapeutic benefit of ICS in COPD patients.

Clinical perspectives

  • Some COPD patients benefit from ICS use; however, the mechanisms are poorly understood. The present study provides information beyond the previously described anti-inflammatory effects on cytokine production.

  • We demonstrate how corticosteroids alter COPD lung macrophage phenotype and function, specifically by increasing the uptake of haemoglobin and apoptotic neutrophils. This may have implications for bacterial growth and tissue damage.

  • These results provide potential mechanisms explaining why ICSs have therapeutic benefit in COPD.

Competing Interests

A.H. has received personal fees from Chiesi. T.S., J.L., R.G., A.B.D., R.S., M.A.M.-F. and S.L. have no conflicts of interest. D.S. personal fees from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, GlaxoSmithKline, Glenmark, Menarini, Mundipharma, Novartis, Peptinnovate, Pfizer, Pulmatrix, Therevance and Verona. JEDC reports grants and personal fees from Aché, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Novartis and Pfizer.

Funding

This work was supported by the National Institute for Health Research Biomedical Research Centre at Wythenshawe Hospital of Manchester University NHS Foundation Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the North West Lung Centre Charity, National Institute for Health Research or the Department of Health. The authors would like to acknowledge the Manchester Allergy, Respiratory and Thoracic Surgery Biobank and the North West Lung Centre Charity for supporting this project.

Author Contribution

A.H., T.S., J.L., R.G., A.B.D. and S.L. performed experimentation. R.S. was the lead surgeon for the study. M.A.M.-F. was the lead histopathologist for the study. A.H., S.L. and D.S. designed the study. All authors were involved in analysis and interpretation of the data, and preparation of the manuscript with major contributions from A.H., S.L. and D.S.

Acknowledgements

We would like to thank the study participants for their contribution.

Abbreviations

     
  • BHI

    brain heart infusion broth

  •  
  • BSA

    bovine serum albumin

  •  
  • CD64

    cluster of differentiation 64

  •  
  • COPD

    chronic obstructive pulmonary disease

  •  
  • FEV1

    lower forced expiratory volume in 1 s

  •  
  • ICS

    inhaled corticosteroid

  •  
  • MDM

    monocyte-derived macrophage

  •  
  • MFI

    median fluoresence intensity

  •  
  • NTHi

    non-typeable Haemophilus influenzae

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PI

    propidium iodide

  •  
  • ROS

    reactive oxygen species

  •  
  • sBHI

    supplemented brain heart infusion broth

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Supplementary data