Post on 09-Jul-2020
Human rhinovirus impairs the innate immune response to bacteria in alveolar
macrophages in COPD
Lydia J Finney¹, Kylie B. R Belchamber¹, Peter S Fenwick¹, Samuel V Kemp¹ ²,
Michael R. Edwards¹, Patrick Mallia¹, Gavin Donaldson¹, Sebastian L Johnston¹,
Louise E Donnelly¹, Jadwiga A Wedzicha¹
¹COPD and asthma section, National Heart and Lung Institute, Imperial College
London, London, UK
²Royal Brompton Hospital, London, UK
Corresponding Author: Dr Lydia J Finney
L.finney@imperial.ac.uk
COPD Research Group
National Heart and Lung Institute
Imperial College
Dovehouse Street
London
SW3 6LY
This article has an online data supplement, which is accessible from this issue's
table of content online at www.atsjournals.org
Total word count: 4,332
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Authorship
LJF designed the study, acquired samples, analysed the data, interpreted results
and drafted the manuscript
KBRB designed experiments, analysed data and interpreted results
PSF designed experiments
SVK acquired samples and interpreted results
MRE contributed to experimental design and conception of the study
PM contributed to experimental design and conception of the study and contributed
to the manuscript
GD analysed data and interpreted results
SLJ designed the study and interpreted results and contributed to the writing of the
manuscript
LED designed the study and experiments, interpreted the results and contributed to
the writing of the manuscript
JAW designed the study, interpreted the results and contributed to the writing of the
manuscript
Grants:
9.7 COPD Exacerbations
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Abstract
Rationale
Human rhinovirus (HRV) is a common cause of COPD exacerbations. Secondary
bacterial infection is associated with more severe symptoms and delayed recovery.
Alveolar macrophages clear bacteria from the lung and maintain lung homeostasis
through cytokine secretion. These processes are defective in COPD. The effect of
HRV on macrophage function is unknown.
Objectives
To investigate the effect of HRV on phagocytosis and cytokine response to bacteria
by alveolar macrophages and monocyte derived macrophages (MDM) in COPD and
healthy controls.
Methods
Alveolar macrophages were obtained by bronchoscopy and MDM by adherence.
Macrophages were exposed to HRV 16 (multiplicity of infection 5), poly I:C 30μg/ml,
interferon (IFN)-β 10μg/ml, IFN-γ 10μg/ml or medium control for 24 hours.
Phagocytosis of fluorescently-labelled Haemophilus influenzae or Streptococcus
pneumoniae was assessed by fluorimetry. CXCL8, IL-6, TNF-α and IL-10 release
was measured by ELISA.
Main Results
HRV significantly impaired phagocytosis of H. influenzae by 23% in MDM (n=37,
p=0.004) and 18% in alveolar macrophages (n=20, p<0.0001) in COPD. HRV also
significantly reduced phagocytosis of S. pneumoniae by 33% in COPD MDM (n=20,
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p=0.0192). There was no effect in healthy controls. Phagocytosis of H. influenzae
was also impaired by poly I:C but not IFN-β or IFN-γ in COPD MDM. HRV
significantly reduced cytokine responses to H. influenzae. The IL-10 response to H.
influenzae was significantly impaired by poly I:C, IFN-β and IFN-γ in COPD cells.
Conclusions
HRV impairs phagocytosis of bacteria in COPD which may lead to an outgrowth of
bacteria. HRV also impairs cytokine responses to bacteria via the TLR3/IFN pathway
which may prevent resolution of inflammation leading to prolonged exacerbations in
COPD.
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At a glance summary
Scientific knowledge of the subject
Respiratory viruses are key triggers of COPD exacerbations. Secondary bacterial
infection is common during an exacerbation and is associated with greater airway
inflammation, a higher symptom burden and impaired recovery. Alveolar
macrophages clear bacteria from the lung by phagocytosis and secrete cytokines
leading to neutrophil recruitment and resolution of inflammation. Macrophage
responses to bacteria are known to be impaired in COPD.
What this study adds to the field
This study is the first to show that human rhinovirus impairs phagocytosis of bacteria
in monocyte-derived macrophages and alveolar macrophages from patients with
COPD. The same effect was not seen in healthy controls. Human rhinovirus also
induced the release of cytokines CXCL8, IL-6, TNF-α and IL-10 from monocyte
derived macrophages, but impaired CXCL8, IL-6, TNF-α and IL-10 release in
response to bacteria. These are possible mechanisms by which human rhinovirus
may lead to an outgrowth of bacteria and delayed recovery from COPD
exacerbations.
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Introduction
Chronic obstructive pulmonary disease (COPD) affects over 174 million people
worldwide, resulting in 3.2 million deaths in 20151. COPD exacerbations are acute
episodes of symptom worsening and are the main cause of hospital admission and
death from COPD2. The majority of exacerbations are caused by respiratory
infections3, commonly respiratory viruses, with human rhinovirus (HRV) being the
most frequently detected4,5. Virus-induced exacerbations are associated with greater
airway inflammation6, more severe symptoms and a delayed recovery time
compared to exacerbations where no virus is detected7.
There is increasing evidence that secondary bacterial infection follows an initial viral
infection during COPD exacerbations4,8. Secondary bacterial infection is associated
with increased dyspnoea, greater airway inflammation and prolonged symptoms
compared to exacerbations where a secondary bacterial infection is not identified4,7,9.
Co-infection also increases the risk of hospital admission and a prolonged length of
stay compared to COPD exacerbations where co-infection is not present10–12. The
interactions between viruses and bacteria during COPD exacerbations remain poorly
understood13.
Alveolar macrophages play a pivotal role in lung defence against invading
pathogens: removing bacteria by phagocytosis14, initiating inflammatory responses
and regulating potentially harmful inflammation15. In COPD, however, the ability of
alveolar macrophages to clear bacteria is impaired16,17. Impaired phagocytosis has
been proposed as a possible mechanism of bacterial infection which could lead to
exacerbations and airway colonisation in COPD14.
Little is known about the effect of HRV infection on macrophage phagocytosis,
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although recent data suggests other respiratory viruses such as influenza and
respiratory syncytial virus may decrease bacterial uptake in human macrophages18.
While previous work has predominantly focused on epithelial cells which are the
primary site of HRV replication19, HRV may induce distinct transcriptome profiles
which are driven towards a pro-inflammatory phenotype in polarized human
macrophages20 and to alter the activation status of alveolar macrophages in a mouse
model of asthma exacerbations21.
We hypothesised that HRV infection may impair phagocytosis of Haemophilus
influenzae and Streptococcus pneumoniae by alveolar macrophages and monocyte-
derived macrophages (MDM) in COPD but not in healthy controls. We postulated
that suppression of phagocytosis by HRV in combination with underlying
macrophage dysfunction, could lead to secondary bacterial outgrowth during COPD
exacerbations.
The aim of this study was to investigate the effect of HRV infection on phagocytosis
of H. influenzae and S. pneumoniae by alveolar macrophages and MDM in COPD
and healthy controls.
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Methods
Participant recruitment
Whole blood was obtained from COPD patients participating in the London COPD
Cohort. Participants represented a range of disease severity. Inclusion criteria were
FEV1/FVC<0.70, smoking history ≥ 10 pack years and able to give informed
consent. Participants were excluded if they were immunosuppressed, had another
significant respiratory disease or active malignancy. Ethics approval was granted by
the London-Hampstead Ethics Committees (REC reference 09/H0720/8). All
participants gave written informed consent.
COPD patients and age matched healthy controls underwent bronchoscopies to
obtain alveolar macrophages. COPD patients were only recruited if they had an
FEV1≥50% due to potential risks of bronchoscopy. The protocol was approved by
Bromley Ethics Committee (REC reference 15/LO/1241). Inclusion criteria for COPD
participants undergoing bronchoscopy were age 40-75 years, FEV1/FVC <0.7,
FEV1≥50% predicted with no other significant respiratory disease. Inclusion criteria
for healthy controls were age 40-75 years, FEV1/FVC ≥ 0.7, <10 pack year smoking
history and no significant respiratory disease. Participants were excluded if they
were immunosuppressed, had active malignancy, any contraindication to
bronchoscopy or were unable to give informed consent. All participants were
recruited when clinically stable with at least 4 weeks symptom free following an
exacerbation to minimise the risk of concomitant virus or bacterial infection
Monocyte-derived macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated by sedimentation
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through a discontinuous Percoll gradient (VWR, Lutterworth, UK) as described
previously22. Monocytes were cultured for 12 days with medium supplemented with
either 2ng/ml GM-CSF (R&D Systems, Abingdon, UK) or 100ng/ml M-CSF (R&D
Systems) to obtain MDM for experimental assays as previously described22.
Alveolar macrophages
Bronchoalveolar lavage was performed and alveolar macrophages obtained by
centrifugation as previously described23. Cells were plated at 1x10⁵ cells per well in
black 96 well plate or 2.5x10⁶ in a 24 well plate (Corning Costar) incubated overnight
prior to experimentation24.
Viral stocks
The type A strain HRV16 was used for viral infection. HRV16 was amplified and
grown in Ohio HeLa cells and the identity of each rhinovirus serotype confirmed
using serotype specific antibody (ATCC), and inactivated by exposure to UV-light for
30 min, as previously described25.
Bacteria
Serotype 9V S. pneumoniae strain (NCTC10692) was grown as previously
described26. Non-typeable H. influenzae strain (NCTC1479) was cultured as
previously described27. Non-opsonized heat-killed (HK) bacteria were generated by
incubation at 65°C for 10 min as described previously27. Bacterial cultures labelled
with Alexa Fluor 488 (Invitrogen, Loughborough, UK) or Alexa Flour 405 NHS ester
(1mg/1ml DMSO, Sigma) as previously described27. Fluorescent bacterial stocks
were sonicated to ensure even distribution of bacteria.
Phagocytosis assay
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In MDM, at 12 days of culture, medium was replaced with RPMI 1640 (no additives).
MDMs were infected with HRV16 at increasing multiplicity of infection (MOI 0.5-10)
for 24 hours or HRV16 (MOI 5) for 3, 6 and 24 hours. For subsequent experiments,
MDM were infected with HRV or UV irradiated HRV at an MOI 5. Alternatively, MDM
were stimulated with Polyinosinic:polycytidylic acid (poly I:C) (Sigma), interferon β
(Sigma), interferon γ (Sigma) diluted in RPMI 1640 media for 24 hours. Alveolar
macrophages were cultured for 24 hours before exposing to HRV (MOI 5) or medium
alone for 24 hours. Following exposure to HRV, poly I:C or interferon, cells were
exposed to Alexa Fluor 488-labelled (Invitrogen) heat killed H. influenzae (1.5x1010
CFU/ml, MOI 1500) or S. pneumoniae (1.7x108 CFU/ml, MOI 17) at 37⁰C for 4 hours.
Extracellular fluorescence was quenched with trypan blue (0.125 mg/ml in phosphate
buffered saline (PBS)). Phagocytic capacity was measured using a fluorimeter
(FLUOstar Optima). Phagocytosis was calculated by subtracting auto-fluorescence
of unstimulated cells. Cell viability was measured using a thiazolyl blue tetrazolium
bromide (MTT) assay as previously described22.
ELISA
Soluble mediators CXCL-8, IL-6, TNF-α, and IL-10 were measured in cell
supernatants using enzyme linked immunosorbent assay (ELISA) according to the
manufacturer’s instructions (R and D systems, UK). The lower limit of detection for
these assays was 31 pg/ml. Plates were read on Spectramax Plus 384 plate reader
using Softmax Pro 6 software.
Confocal microscopy
Confocal microscopy was performed to confirm whether HRV16 infected alveolar
macrophages. Cells were infected with PKH26 (Sigma) labelled HRV16 (MOI 10) or
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sham infected with PKH26 and PBS for 24 hours before performing a phagocytosis
assay with Alexa Fluor 405-labelled heat killed H. Influenzae (1.5x1010 CFU/ml) or
(1.7x108 CFU/ml). Cells were fixed with 4% (w/v) paraformaldehyde and methanol
before staining the cytoplasm with 20M Cell tracker green CMFDA (Thermo Fisher,
Loughborough, UK) and the nucleus with DRAQ5 (Thermo fisher). Images were
taken on a Zeiss LSM-510 inverted confocal microscope and analysed using Fiji
software.
Statistics
Paired measurements were measured with Wilcoxon’s signed-rank test and unpaired
data analysed using Mann-Whitney U test. In the case of repeated measures from
the same donor, data was analysed with Friedman’s test with Dunn’s post-test
corrected for multiple comparisons since if multiple cytokines have been examined,
there is a risk of false discovery. Correlations between datasets were examined
using Spearman’s rank correlation coefficient. All data were analysed using
GraphPad PRISM v7 (GraphPad Software Inc, San Diego, USA). Differences were
considered significant if p<0.05. Data are presented as median (interquartile range)
unless otherwise stated.
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Results
Patient demographics
Alveolar macrophages were obtained from 20 COPD patients with an FEV1 ≥ 50%
predicted and 16 healthy controls. COPD participants had a significantly lower FEV1
(litres), FEV1% predicted and FEV1/FVC compared to healthy controls and a
significantly greater smoking history. COPD patients were also significantly older
than healthy controls (Table 1). None of the participants undergoing bronchoscopy
were known to be colonised with bacteria based on microbiological culture. There
was no significant difference between bronchoalveolar lavage cell counts
(Supplement Table 1). MDM were obtained from a total of 37 COPD patients to
include a spectrum of disease severity, including patients with more severe disease
(Table 2).
HRV16 impairs phagocytosis of bacteria in MDM from COPD participants but
not healthy controls
To investigate the effect of HRV16 on phagocytosis of bacteria, HRV16 was added
to MDM from COPD patients for 24 hours prior to phagocytosis of bacteria. HRV16
significantly impaired phagocytosis of H. Influenzae at MOI 2.5 (3.14 RFUx10³), MOI
5 (3.00 RFUx10³) and MOI 10 (3.03 RFUx10³) compared to media control (4.11
RFUx10³) p=0.0003, Figure 1A). A similar effect was observed with S. pneumoniae
(8.96 RFUx10³ media control) with significant reductions in uptake following
exposure of cells to MOI 2.5 (6.32 RFUx10³), MOI 5 (6.12 RFUx10³) and 10 (5.51
RFUx10³) respectively p=0.0006 (Figure 1B) with no effect on cell viability
(Supplement figure 1A, B). By contrast, HRV16 did not impair phagocytosis of latex
beads (supplement figure 1C, D) suggesting that this effect is specific to bacterial
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pathogens. Of note, HRV16 at MOI 5 did not significantly impair phagocytosis of H.
influenzae in MDM from healthy controls (supplement figure 2A, B).
A time course analysis was performed to investigate if the reduction of phagocytic
capacity by HRV16 was time-dependent, therefore MDM were incubated with the
virus for up to 24 hours. Under these conditions, virus significantly reduced
phagocytosis of H. influenzae at 6 hours (1.66 RFUx10³) and 24 hours (2.02
RFUx10³) compared to medium control (3.51 RFUx10³) p=0.002, Figure 1C). HRV16
also significantly reduced phagocytosis of S. pneumoniae after 6 hours (1.63
RFUx10³) and 24 hours (2.20 RFUx10³) compared to media control (6.00 RFUx10³)
p=0.0004 (Figure 1D).
These time course experiments suggested that the effect of HRV16 on phagocytosis
required incubation of between 6-24 hours, suggesting the effect is either dependent
on viral replication or a secondary mediator. To further investigate whether the effect
of HRV on phagocytosis may be related to viral replication, MDM were infected with
either HRV16 (MOI 5), UV-irradiated HRV16 (MOI 5) or media control for 24 hours
before performing a phagocytosis assay. Phagocytosis of H. influenzae by COPD
MDM was significantly reduced by live HRV16 (1.85 RFUx10³) but not UV-irradiated
HRV16 (2.51 RFUx10³) compared to media control (2.14 RFUx10³) p=0.0014,
(Figure 1E). The same effect was seen with S. pneumoniae (Figure 1F).
The effect of macrophage phenotype on the response to HRV16 mediated
suppression of phagocytosis was investigated using the MDM model. Monocytes
were differentiated in the presence of either GM-CSF or M-CSF to model the effect
of HRV on classically activated (M1) macrophages and alternatively activated (M2)
macrophages respectively28.
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HRV16 significantly impaired phagocytosis of H. influenzae in both GM-CSF
differentiated MDM (HRV16 2.25 RFUx10³ versus media control 2.72 RFUx10³
p=0.001 Figure 2A) and M-CSF differentiated MDM HRV16 (2.96 RFUx10³) versus
medium control (3.58 RFUx10³) p<0.0001, (Figure 2C). HRV16 also significantly
impaired phagocytosis of S. pneumoniae in GM-CSF differentiated MDM (Figure 2B)
and M-CSF differentiated MDM HRV16 (Figure 2D). These data suggest that the
phagocytic response of macrophages to HRV16 infection is consistent across
macrophage phenotypes.
Effect of HRV16 on phagocytosis of H. influenzae and S. pneumoniae in
alveolar macrophages from COPD patients and healthy controls
Alveolar macrophages from COPD patients and healthy controls were infected with
HRV16 (MOI 5) for 24 hours before performing a phagocytosis assay. HRV16
significantly impaired phagocytosis of H. influenzae by alveolar macrophages in
COPD patients - HRV16 (0.84 RFUx10³) versus (1.17 RFUx10³) medium control
p<0.001 Figure 3A) but did not impair phagocytosis of S. pneumoniae (Figure 3B).
HRV16 did not impair phagocytosis of H. influenzae or S. pneumoniae in alveolar
macrophages from healthy controls (Figure 3C, D). Inhaled corticosteroid use did not
attenuate the phagocytic response to HRV in the COPD group (supplement figure 3).
Confocal microscopy confirms HRV16 enters macrophages
There has been debate as to whether HRV is able to enter and replicate within
alveolar macrophages29. Therefore, confocal microscopy of alveolar macrophages
from a COPD participant was performed using PKH26 labelled HRV or sham
infection to investigate whether HRV16 entered alveolar macrophages. Cells infected
with PKH26-labelled HRV showed HRV within the cytoplasm of the cell separate to
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bacterial uptake, but the virus did not appear to enter the nucleus of the cell (Figure
4.1). Sham infection showed PKH26 uptake in phagosomes with bacteria only (figure
4.2).
Phagocytosis of bacteria is impaired in alveolar macrophages and MDM in
COPD
Baseline phagocytic capacity for H. influenzae and S. pneumoniae was assessed in
alveolar macrophages and MDM from both healthy controls and COPD patients, to
confirm previous studies showing impairment of phagocytosis was impaired in the
COPD. Phagocytosis of H. influenzae and S. pneumoniae was significantly impaired
in alveolar macrophages from COPD patients compared to healthy controls: H.
influenzae (3.31 RFUx10³) healthy controls versus (1.38 RFUx10³) COPD p=0.0008;
and S. pneumoniae (4.86 RFUx10³) healthy controls versus (1.86 RFUx10³) COPD
p=0.0002 (supplement figure 4A and B). Phagocytosis of H. influenzae was also
suppressed in MDM from patients with COPD compared to healthy controls p=0.004.
However, the same effect was not seen in MDM with S. pneumoniae (supplement
figure 4C and D).
There was a correlation between alveolar macrophage phagocytosis of H. influenzae
and FEV1% predicted r=0.6787, p=0.002 and S. pneumoniae with FEV1% predicted
r=0.5947, p=0.0273 in the COPD group. A relationship was also seen between FEV1
(litres) and phagocytosis of H. influenzae r=0.5108, p=0.0303 but not S. pneumoniae
(supplement figure 5 A-D). There was no relationship between inhaled corticosteroid
use or current smoking status and baseline phagocytic capacity (Supplement figure
6A-D).
Comparison of the phagocytic response between AM and MDM from the same
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subject showed a correlation with respect to phagocytosis of H. influenzae r=0.7922,
p<0.0001 (Figure 5A) and S. pneumoniae r=0.539, p=0.0210 (Figure 5B).
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HRV16 induces pro-inflammatory and anti-inflammatory cytokine release from
MDM
MDM were infected with HRV16 for 24 hours and CXCL8, TNF-α, IL-6 and IL-10
were measured by ELISA. Increasing concentrations of HRV16 induced the
production of CXCL8 (media control 4.41 ng/ml versus HRV MOI 5 20.26 ng/ml and
MOI 10 24.65 ng/ml respectively p<0.0001), IL-6 (media control 0.96 ng/ml versus
MOI 5 2.66 ng/ml and MOI 10 3.61 ng/ml p=0.0002), TNF-α (media control 5.32
pg/ml versus MOI 5 52.7 pg/ml and MOI 10 107.8 pg/ml p=0.012) and IL-10 (media
control 20.62 pg/ml versus MOI 5 41.12 pg/ml and MOI 10 56.85 pg/ml p=0.0002
(Figure 6).
HRV16 impairs the cytokine response to bacteria in MDM from COPD
participants
MDM were infected with HRV16 for 24 hours or media control prior to infecting with
H. influenzae for 4 hours. CXCL8, TNF-α, IL-6 and IL-10 were measured using
ELISA. HRV16 significantly impaired release of CXCL8 (media control 4.43 ng/ml
versus MOI 5 1.8 ng/ml and MOI 10 2.91 ng/m p=0.0002), TNF-α (media control
23.39 ng/ml versus MOI 5 11.41 ng/ml and MOI 10 9.61 ng/ml p=0.002), IL6 (media
control 2.49 ng/ml versus MOI 10 0.974 ng/ml, p=0.0110) and IL-10 (media control
1.82 ng/ml versus HRV16 MOI 5 0.63 ng/ml vs MOI 10 0.42 ng/ml, p=0.002), by
MDM in response to H. influenzae compared to media control (Figure 6).
Phagocytosis of H. influenzae is reduced by Poly I:C but not Type I or Type II
interferons in MDM
In airway epithelial cells, HRV is taken up by receptor mediated endocytosis. Viral
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RNA then binds to TLR3, TLR7 and TLR8 leading to transcription of pro-
inflammatory cytokines as well as type I, II and III interferons30. To explore potential
pathways by which HRV16 may inhibit macrophage phagocytosis of bacteria, MDM
were stimulated with either the TLR3 agonist poly I:C, IFN-γ or IFN-β for 24 hours
before performing a 4-hour phagocytosis assay with H. influenzae.
Poly I:C impaired phagocytosis of H. influenzae in a concentration-dependent
manner (media control 3.72 RFUx10³) versus poly I:C 300 µg/ml 1.91 RFUx10³
p=0.0002,) and impaired IL-10 response to H. influenzae (media control 0.40 ng/ml
versus poly I:C 300 µg/ml 0.12 ng/ml). IFN-γ (100 μg/ml) and IFN-β (10 μg/ml) both
impaired IL-10 response to H. influenzae (media control 0.60 ng/ml versus IFN-γ
0.35 ng/ml p=0.04, media control 1.32 ng/ml versus IFN-β 0.31 p=0.008 respectively)
but in contrast to poly I:C, did not impair phagocytosis of bacteria (Figure 7).
In healthy controls, phagocytosis of H. influenzae was not significantly impaired by
HRV16, poly I:C, IFN-β or IFN-γ. IL-10 response to H. influenzae was impaired by
HRV16 (media control 0.84 ng/ml versus 0.24 ng/ml p=0.0041) but not poly I:C, IFN-
β, or IFN-γ (supplement figure 7).
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Discussion
This is the first study to show that human rhinovirus impairs phagocytosis of bacteria
by alveolar macrophages and MDM from patients with COPD. HRV16 also impaired
cytokine CXCL8, IL-6, TNF-α and IL-10 responses to bacteria which may be
mediated via the TLR3/ IFN pathway. This study clearly demonstrates that HRV16
alters the innate immune response in COPD cells.
Respiratory viruses are key triggers of COPD exacerbations7 and HRV has been
identified in up to 60% of COPD exacerbations with viral load being greatest at
exacerbation onset.7 Studies of the time course of HRV induced COPD
exacerbations suggest an initial viral infection is followed by an outgrowth of bacteria
with a peak in bacterial load two weeks after symptoms 7,9. Dual infection with HRV
and bacteria during an exacerbation is also associated with more severe symptoms,
greater airway inflammation and a greater decline in FEV1 compared to
exacerbations where only one pathogen is present4,8,9, suggesting a synergistic effect
between HRV and bacteria leading to bacterial outgrowth.
Alveolar macrophages are the primary phagocytic cell of the lung, where their key
roles are phagocytosis of bacteria31, clearance of apoptotic cells32 and modulation of
the inflammatory environment through release of cytokines and other mediators15.
Alveolar macrophages comprise multiple phenotypes, which are highly plastic and
able to adapt to their microenvironment33,34. However, several of these functions are
suppressed in COPD35 and recent studies have shown that alveolar macrophages do
not conform to these phenotypes in COPD36. It is therefore important to study
multiple macrophage phenotypes to determine whether similar defects exist in the
laboratory setting.
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In this study, HRV16 impaired phagocytosis of S. pneumoniae in MDM, and H.
influenzae in both MDM and alveolar macrophages from patients with COPD. This is
supported by Oliver et al who found HRV16 impaired phagocytosis of Escherichia
coli particulates in alveolar macrophages from 3 individuals37. However, important
strengths of the current study are the use of whole bacteria S. pneumoniae and H.
influenzae which are two of the most commonly identified bacteria in COPD
exacerbations4,10,38,39. Studying the effect of HRV on phagocytosis of H. influenzae is
particularly clinically relevant because of increasing evidence of an interaction
between HRV and H. influenzae in COPD exacerbations8,38,40. Secondly, alveolar
macrophages and MDM were obtained from a well characterised group of COPD
participants and healthy controls. This has revealed a distinct difference in the effect
of HRV infection on macrophage phagocytosis, with no significant effect of HRV on
phagocytosis of bacteria in healthy controls. This suggests that HRV enhances a
specific defect in COPD that may increase susceptibility to secondary bacterial
infection. These data suggest that viral suppression of macrophage phagocytic
capacity is a possible mechanism for bacterial outgrowth during COPD
exacerbations.
Viral suppression of phagocytosis was seen in MDM differentiated in both GM-CSF
and M-CSF, suggesting that HRV did not impair phagocytosis by simply inducing
functional plasticity. This impairment required live HRV and suggests that
suppression of phagocytosis may be either an effect of viral replication, as a result of
viral protein transcription, or the effect of a secondary intermediary. Using PKH26-
labelled HRV16, we showed that the virus is located in the cytoplasm of alveolar
macrophages: however, further work is needed to elucidate the mechanism of
reduction in phagocytosis and any relationship to viral replication.
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While in vitro studies using BAL or MDM clearly show HRV may infect, replicate and
induce cytokine responses29,37,41 whether or not this occurs in vivo remains
controversial. In support, HRV has also been shown to co-localize with CD68+
macrophages in a mouse infection model, suggesting HRV directly infects
macrophages in vivo21. Furthermore, in experimentally infected humans, HRV load in
BAL fluid is positively associated with CD68+ cells, suggesting macrophages may be
an important site for HRV replication42. Further work is needed to confirm if
macrophages are infected by HRV during COPD exacerbations. While these
experiments are beyond the scope of the present study, COPD patients
experimentally infected with HRV show increased lung bacterial burden40 consistent
with the proposed mechanism of prior HRV infection, limiting the ability of airway
macrophages to control bacteria outgrowth in COPD.
Defective phagocytosis of bacteria by alveolar macrophages in COPD has been
proposed as a possible mechanism for bacterial colonisation and susceptibility to
exacerbation17. This study supports previous findings, but also demonstrated a
relationship between baseline phagocytic capacity for H. influenzae by alveolar
macrophages and FEV1% predicted, suggesting phagocytic impairment is also
related to disease severity. This is in agreement with work by Berenson et al who
also found a relationship between FEV1 and phagocytosis of H. influenzae and
Moraxella catarrhalis in alveolar macrophages43,44.
Obtaining alveolar macrophages by bronchoalveolar lavage can be difficult in severe
COPD or during exacerbations due to increased risk of bronchoscopy45. This study is
the first to show a correlation between phagocytic capacity in paired alveolar
macrophages and MDM (figure 5), suggesting MDM could be used as a model of
alveolar macrophage phagocytosis. These findings contrast with Berenson et al who
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found no relationship between phagocytosis by alveolar macrophages and MDM16,31.
These differences may be because MDM were differentiated using GM-CSF and M-
CSF in this study, whereas Berenson et al did not 31. MDM differentiated with GM-
CSF have previously been shown to express cell surface receptors similar to
alveolar macrophages46 and are used by other groups18,27,28,47. MDM have the
advantage of being an accessible and high yield method for obtaining
macrophages46 with the potential to evaluate new therapeutic targets in a minimally
invasive manner. They are also not affected by the low yield and contamination
issues seen with sputum macrophages48. However, further work is needed to confirm
that MDM can be used as a model of alveolar macrophages in more severe disease.
A key function of alveolar macrophages is producing pro-inflammatory cytokines to
recruit neutrophils and monocytes to the site of injury49. In this study, HRV16 induced
pro-inflammatory cytokines CXCL8, IL-6 and TNF-α release from MDM. This is in
agreement with previous work showing HRV16 and HRV1b induce CXCL8 and TNF-
α from MDM and AM from atopic individuals and healthy controls37,50. In contrast to
Oliver et al37 however, HRV16 induced the anti-inflammatory cytokine IL-10, but at
much lower levels than CXCL8, IL-6 and TNF-α, suggesting that HRV induces a
predominantly pro-inflammatory response in COPD MDM. This pro-inflammatory
environment may contribute to the increased airway inflammation and sputum
neutrophilia seen during HRV induced exacerbations51.
The ability of macrophages to produce pro-inflammatory cytokines such as CXCL8,
IL-6 and TNF-α is important for clearing bacteria from the lung. The anti-
inflammatory cytokine IL-10 is produced in parallel, playing a crucial role in
maintaining homeostasis and preventing excessive inflammation which may damage
the host52. HRV16 has previously been shown to impair pro-inflammatory cytokine
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responses to the bacterial product lipopolysaccharide (LPS) in alveolar
macrophages37. We found exposure of MDM to HRV16 reduced CXCL8, IL-6, TNF-α
and IL-10 responses to H. influenzae: suggesting that HRV has a globally
suppressive effect on the innate immune response to bacteria, which may lead to
increased susceptibility to secondary bacterial infection and prolonged COPD
exacerbations. This effect cannot be attributed to cell death as our data showed that
HRV did not significantly reduce cell viability.
Alveolar macrophages are also the main producers of type I interferons during
pulmonary viral infection53 with several in vitro studies showing that HRV induces
type I and type II IFNs in AM and MDM54,55. Type I interferons act in an autocrine and
paracrine manner via interferon stimulated genes (ISGs) to achieve early control of
viral replication and recruit monocytes, Th1 and NK cells to areas of infection56.
However, the role of type I interferons in bacterial infection is controversial, as they
appear to increase susceptibility to some bacterial infections but provide protection
against others56. In this study, the TLR3 agonist poly I:C impaired phagocytosis of H.
influenzae and reduced production of CXCL8, TNF-α, IL-6 and IL-10 in response to
H. influenzae. In contrast, IFN-β and IFN-γ did not impair phagocytosis of bacteria
but did inhibit the IL-10 response in MDM. These findings suggest HRV has several
inhibitory effects on the macrophage response to bacteria, with cytokine inhibition
and phagocytosis suppression being mediated by two distinct mechanisms. Further
work to understand these mechanisms offers the potential to develop new
therapeutic targets for use in COPD exacerbations, such as the use of JAK/STAT
inhibitors to modulate the TLR3/ interferon pathway57.
Few studies have examined the role of IL-10 in COPD: however, Berenson et al
found the IL-10 response to LOS, which is one of the outer membrane components
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of non-typeable H. influenzae, was impaired in alveolar macrophages from COPD
patients compared to healthy controls16. IL-10 deficiency in mice has also been
associated with excessive inflammation in the presence of micro-organisms58. This
could be important in COPD as it is a disease characterised by excessive airway
inflammation and airway bacterial colonisation59. These findings suggest that HRV
may impair the IL-10 response to H. influenzae via the TLR3/IFN pathway with a
dysregulation of inflammation. This could result in excessive and sustained airway
inflammation leading to prolonged exacerbations in COPD. Further work is needed
to examine the role of IL-10 in COPD and whether there is a therapeutic role for IL-
10 in resolution of exacerbations.
Conclusions
Human rhinovirus suppressed phagocytosis of bacteria by alveolar macrophages
and monocyte derived macrophages in COPD patients but not healthy controls. This
may be due to the enhancement of an existing phagocytic defect in COPD
macrophages. We propose a dual-hit hypothesis, where baseline macrophage
phagocytic dysfunction in combination with further impairment by human rhinovirus
leads to an outgrowth of bacteria and exacerbations in COPD. Human rhinovirus
also further reduced the cytokine response to bacteria in COPD: suggesting a
globally suppressive effect which may prevent resolution of inflammation, leading to
prolonged exacerbations in COPD.
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Acknowledgements
We would like to thank all members of the London COPD Cohort and healthy
volunteers who have participated in this study.
The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is
part supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and
BBSRC (grant BB/L015129/1)
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Figure Legends
Figure 1. The effect of human rhinovirus 16 (HRV) on phagocytosis of bacterial
pathogens by monocyte derived macrophages (MDM) from COPD patients.
MDM were infected with HRV at increasing multiplicity of infection (MOI) for 24 hours
followed by exposure to fluorescently labelled H. influenzae (panel A) or S.
pneumoniae (panel B). A time course of HRV (MOI 5) infection of 3, 6 or 24 hours,
followed by phagocytosis of H. influenzae (panel C) or S. pneumoniae (panel D).
MDM were infected with either UV irradiated HRV16 (MOI 5), HRV16 (MOI 5) or
media control, followed by phagocytosis of H. influenzae (panel E) or S. pneumoniae
(panel F). Phagocytosis was assessed by fluorimetry. Data are presented in relative
fluorescent units (RFU) where each point represents an individual subject with
median and interquartile range. Analysis was performed using Friedman’s test with
Dunn’s post-test where *= p<0.05, **=p<0.01 and ***=p<0.001
Figure 2. The effect of human rhinovirus 16 (HRV) on phagocytosis of bacterial
pathogens by monocyte derived macrophages stimulated with GM-CSF or M-
CSF from COPD patients. Monocytes were cultured in media supplemented with
GM-CSF (panel A and B) or M-CSF (panel C and D) for 12 days prior to
experimentation. MDM were infected with HRV (multiplicity of infection 5) for 24
hours before exposure to fluorescently labelled H. influenzae (panel A and C) or S.
pneumoniae (panel B and D). Phagocytosis was measured using fluorimetry. Data
are presented in relative fluorescent units (RFU) where each point represents an
individual subject. Analysis was performed using Wilcoxon’s signed-rank test where
*=p<0.05 and ***=p<0.001.
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Figure 3. The effect of human rhinovirus 16 (HRV) on phagocytosis of bacterial
pathogens by alveolar macrophages from COPD patients or healthy controls.
Alveolar macrophages from COPD patients (Panels A, B), or healthy controls
(Panels C and D) were exposed to HRV (MOI 5) for 24 hours before exposure to
fluorescently labelled H. influenzae (panel A and C) or S. pneumoniae (panel B and
D). Phagocytosis was measured by fluorimetry. Analysis was performed using
Wilcoxon signed-rank test where ***=p<0.001.
Figure 4. Confocal microscopy. 1. Alveolar macrophages from a patient with
COPD were infected with PKH26 labelled HRV (panel B, red) for 24 hours before
exposure to Alexa 405 labelled H. influenzae (panel A, blue) for 4 hours. Cells were
fixed and cytoplasm labelled with cell tracker green (panel C, green) and nucleus
labelled with DRAQ5 (Panel D, grey). Images were taken at x63 magnification. Panel
E shows a composite image. Panel F shows a blank panel with a scale bar.
2. Alveolar macrophages from a patient with COPD were exposed to a sham
infection of PKH26 with phosphate buffered saline and media (panel B, red) for 24
hours before exposure to Alexa 405 labelled H. influenzae (panel A, blue) for 4
hours. Cells were fixed and cytoplasm labelled with cell tracker green (panel C,
green) and nucleus labelled with DRAQ5 (Panel D, grey). Images were taken at x63
magnification. Panel E shows a composite image. Panel F shows a blank panel with
a scale bar.
36
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Figure 5. Comparison of phagocytosis of bacterial pathogens by alveolar
macrophages (AM) and monocyte derived macrophages (MDM) from the same
individual. AM and MDM were exposed to fluorescently labelled H. influenzae
(panel A) or S. pneumoniae (panel B) and phagocytosis measured by fluorimetry.
Data are presented in relative fluorescent units (RFU) where each point represents
an individual subject. Analysis performed using Spearman’s rank test.
Figure 6. The effect of HRV16 on cytokine release by MDM from COPD
patients. GM-CSF (panels A – F) or M-CSF (panels G and H) differentiated MDM
were infected with HRV16 at increasing MOI for 24 hours and supernatants
removed. CXCL8 (panel A), TNF-α (panel C), IL-6 (panel E) and IL-10 (panel G)
were measured in supernatants using ELISA. Cells were then infected with H.
influenzae for 4 hours and supernatants removed. CXCL8 (panel B), TNF-α (panel
D), IL-6 (panel F) and IL-10 (panel H) were measured in supernatants using ELISA.
Data are presented in ng/ml or pg/ml where each point represents an individual
subject with median and interquartile range. Analysis was performed using Friedman
test with Dunn’s post-test where *=p<0.05 and **=p<0.01
Figure 7. The effect of poly I:C, interferon β and interferon γ on phagocytosis
and IL-10 response to H. influenzae by MDM from COPD patients. To assess the
effect of the interferon pathway on phagocytosis and IL-10 production MDM were
exposed to poly I:C at increasing concentrations (panel A and B), interferon γ (10
ng/ml, panel C and D), interferon β (10 ng/ml, panel E and F), media control or
human rhinovirus (MOI 5) for 24 hours and then exposed to fluorescently labelled H.
37
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influenzae. Supernatants were removed and IL-10 measured by ELISA (panels B, D
and F). Phagocytosis was measured by fluorimetry (panels A, C and E). Data are
presented are in relative fluorescent units (RFU) where each point represents an
individual subject with median and interquartile range. Analysis was performed using
Friedman’s test with Dunn’s post-test where *= p<0.05 and **=p<0.01.
38
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773
Table 1. Demographics for COPD patients and healthy controls participating in the
bronchoscopy study. Data are presented as median (interquartile range) unless
otherwise stated
Healthy (n=16) COPD (n=20) P value
Age in years (IQR) 58 (55 – 61) 65 (61 – 68) P=0.0044
Male n (%) 10 (62.5%) 13 (68%) ns
Current smokers n (%) 0 (0%) 7 (36%) ns
FEV1 (L) 2.54 (1.86 – 4.10) 1.75 (0.90 – 3.09) P<0.0001
FEV1% predicted 88 (80 – 117) 61 (50 – 93) P<0.0001
FEV1/FVC ratio 0.74 (0.7-0.83) 0.56 (0.37-0.66) P<0.0001
Smoking pack years 0 (0-8) 40 (10-100) P<0.0001
Exacerbations in last year NA 2 (0-4) NA
Inhaled corticosteroids n (%) 0 10 (50%) NA
39
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775
776
Table 2. Demographics for COPD patients providing whole blood for monocyte
derived macrophages. Data are presented as median (interquartile range) unless
otherwise stated
Demographic data Number
Age in years median (IQR) 71 (66.5 – 76)
Male n (%) 28 (75.7%)
FEV1 litres median (IQR) 1.66 (1.24 – 2.03)
FEV1 % predicted median (IQR) 60 (49 – 70)
Exacerbations median (IQR) 2 (0 – 4)
Frequent exacerbators n (%) 19 (51.4%)
GOLD stage n (%)
- 1
- 2
- 3
- 4
3 (8.1%)
22 (59.5%)
10 (27.0%)
2 (5.4%)
Current smoker n (%) 7 (18.9%)
Inhaled corticosteroid use n (%) 32 (86.5%)
LAMA use n (%) 34 (91.8%)
LABA use n (%) 37 (100%)
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Figures
Figure 1
41
Figure 2
42
Figure 3
43
Figure 4
44
ED
CBA
A B C
D E
1
2
Figure 5
45
0 2 4 6 80
2
4
6
Phagocytosis of H. influenzaeby MDM (RFU x 103)
Phag
ocyt
osis
ofH
. inf
luen
zae
by A
M (R
FU x
103 )
R=0.792p<0.0001
0 2 4 6 8 100
2
4
6
8
Phagocytosis of S. pneumoniaeby MDM (RFU x103)
Phag
ocyt
osis
ofS
. pne
umon
iae
by A
M (R
FU x
103 )
R=0.539p=0.021
BA
Figure 6
46
0 1 5 100
10
20
30
40
HRV MOI
CXCL
8 ng
/ml
** **
0 1 5 100
2
4
6
HRV MOICX
CL8
ng/m
l
***
0 1 5 100
1
2
3
4
HRV MOI
IL10
ng/
ml
* **
0 1 5 100
2
4
6
HRV MOI
IL6
ng/m
l
* *
0 1 5 100
1
2
3
4
5
HRV MOI
IL6
ng/m
l
**
0 1 5 100
20
40
60
80
100
HRV MOI
IL10
pg/
ml
* **
0 1 5 100
10
20
30
40
HRV MOI
TNF-
n
g/m
l
** *
0 1 5 100
50
100
150
200
HRV MOI
TNF-
n
g/m
l
**
HG
FE
DC
BA
Figure 7
47
control 3 30 300 HRV0
1
2
3
4
5
poly I:C g/ml
Phag
ocyt
osis
ofH
.influ
enza
e(R
FU x
103 )
** **
0 3 30 3000.0
0.5
1.0
1.5
poly I:C g/mlIL
10 n
g/m
l
*
control IFN HRV0
2
4
6
8
Exposure
Phag
ocyt
osis
ofH
.influ
enza
e(R
FU x
103 )
*
control IFN HRV0
2
4
6
8
10
Exposure
Phag
ocyt
osis
ofH
.influ
enza
e(R
FU x
103 )
**
control 1 10 100 HRV0.0
0.2
0.4
0.6
0.8
1.0
[IFN g/ml]
IL10
ng/
ml
** *
control0.1 1 10 HRV0.0
0.5
1.0
1.5
2.0
2.5
IL10
ng/
ml
** **
[IFN g/ml]
FE
DC
BA