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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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

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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

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2

3

4

HRV MOI

IL10

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* **

0 1 5 100

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HRV MOI

IL6

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* *

0 1 5 100

1

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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