1 Bacterial load and defective monocyte-derived macrophage bacterial phagocytosis in biomass-smoke COPD Baishakhi Ghosh 1,3 , Akshay H Gaike 2,5 , Kanchan Pyasi 1 , Bill Brashier 1 , Vandana V Das 1 , Jyoti D Londhe 1 , Sanjay Juvekar 5 , Yogesh S Shouche 2 , Louise E Donnelly 4 , Sundeep S Salvi 1* , Peter J Barnes 4 1 Chest Research Foundation, Pune, India 2 National Centre for Microbial Resource, National Centre for Cell Sciences, Pune, India 3 Faculty of Health and Biomedical Sciences, Symbiosis International University, Pune, India 4 National Heart and Lung Institute, Imperial College London, London, United Kingdom 5 Vadu Rural Health Program, KEM Hospital Research Centre, Pune, India Corresponding Author: Dr. Sundeep S Salvi, Chest Research Foundation, Survey No. 15, Marigold Complex, Kalyani Nagar, Pune – 411014, India 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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Bacterial load and defective monocyte-derived macrophage bacterial phagocytosis in
biomass-smoke COPD
Baishakhi Ghosh1,3, Akshay H Gaike2,5, Kanchan Pyasi1, Bill Brashier1, Vandana V Das1,
Jyoti D Londhe1, Sanjay Juvekar5, Yogesh S Shouche2, Louise E Donnelly4, Sundeep S
Salvi1*, Peter J Barnes4
1Chest Research Foundation, Pune, India
2National Centre for Microbial Resource, National Centre for Cell Sciences, Pune, India
3Faculty of Health and Biomedical Sciences, Symbiosis International University, Pune, India
4National Heart and Lung Institute, Imperial College London, London, United Kingdom
5Vadu Rural Health Program, KEM Hospital Research Centre, Pune, India
Corresponding Author:
Dr. Sundeep S Salvi,
Chest Research Foundation,
Survey No. 15, Marigold Complex, Kalyani Nagar, Pune – 411014, India
activity of MDMs from tobacco-smoke exposed subjects (S-COPD and HS) negatively
correlated with increased load of Strep. pneumoniae and H. influenzae in induced sputum
(Figure S23 & S24).
Relationship between bacterial phagocytosis by MDMs and spirometric lung function
indices
Having shown differences in PPBs phagocytic activity by MDMs and its association with
bacterial load in airways, we wanted to determine if these differences were associated with
changes in lung function parameters. We observed that in both biomass-smoke and tobacco-
smoke exposed subjects, the MDMs phagocytosis of Strep. pneumoniae and H. influenzae
was positively associated with spirometric lung function indices (P<0.05, Figure S25 – S36).
Relationship between bacterial phagocytosis of MDMs and demographic characteristics
We also investigated whether there is any relationship between bacterial phagocytic activity
of MDMs and demographic characteristics. We found that age was not associated with
MDMs phagocytic activity across all subjects (P>0.05) (Table S9). Both biomass and
tobacco-smoke exposure history were strongly positively associated with MDMs
phagocytosis of Strep. pneumoniae (Table S9). However, there was a weak correlation with
MDMs phagocytosis for H. influenzae (Table S9).
Relationship between bacterial phagocytosis by MDMs and quality of life of COPD
subjects
We investigated the relationship between the MDMs phagocytic activity for PPBs with the
quality of life of COPD subjects. For BMS-COPD, there was a significant negative
association between the total CAT score and MDMs phagocytosis of Strep. pneumoniae (r= -
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0.444, P=0.034) and MDMs phagocytic activity for H. influenzae (r= -0.416, P=0.048
(Figure S37). Similar significant negative association was observed between the total SGRQ
score with MDMs phagocytosis of Strep. pneumoniae (r= -0.453, P=0.029), and H.
influenzae (r=0.579, P=0.007) (Figure S39).
A similar relationship was also observed for S-COPD subjects, where there appeared
to be a significant negative association between the total CAT and SGRQ score and the
phagocytic activity of MDMs for Strep. pneumoniae or H. influenzae (Figure S38 & S40).
Viability assay
It was possible that the reduced phagocytic responses observed could be due to bacteria
induced cell death. In order to investigate this, MTT assays to measure cell viability were
performed. Throughout the experimental conditions the viability of MDMs was ≥90% for all
samples (Figure 8).
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DISCUSSION
Macrophages are key protective innate immune cells that guard against invasion of PPBs.
The S-COPD subjects harbour increased load of PPBs such as Strep. pneumoniae, H.
influenzae, M. catarrhalis, and P. aeruginosa in the lower airways, that are likely to
contribute in recurrent infections in the respiratory tract [7][9][11]. The burden of these PPBs
in the respiratory tract of S-COPD subjects were found to be associated with decline in lung
function indices and impairment in clearance of PPBs by the alveolar macrophages that is
reflected in the response of MDMs as well [14][15][17][34]. In this study, we too observed
that the respiratory tract of S-COPD subjects from a rural Indian population harbour similar
PPBs, which are associated with defective phagocytic activity of MDMs and decline in
spirometric lung function indices.
Biomass-smoke has been suggested to be greater risk factor for COPD globally than
tobacco-smoke because of the sheer numbers of people exposed to biomass-smoke in the
world [5]. It is possible that long-term exposure to biomass-smoke is associated with a
similar defect in the innate immunity as observed in S-COPD. We report here for the first
time in BMS-COPD subjects, a similar association of PPBs colonization in the respiratory
tract with the defect in phagocytic activity of MDMs and a reduction in lung function.
We found that the bacterial loads of Strep. pneumoniae and H. influenzae in sputum
samples were similar in both BMS-COPD and S-COPD subjects. Consequently, phagocytic
activity of MDMs from BMS-COPD subjects were investigated and compared with healthy
controls (H-NS, HS, H-BMS) and S-COPD subjects. Phagocytic activity of MDMs from
BMS-COPD subjects were not significantly altered in comparison to S-COPD subjects, but
were much reduced compared to healthy controls. This suggests that similar mechanisms
relating to defects on the innate immune response may apply in both BMS-COPD and S-
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COPD. There is no doubt that biomass-smoke increases particular uptake in alveolar
macrophages and this may contribute to defective phagocytosis [35][36][37]. However, we
report reduced phagocytosis by MDMs that have not been exposed to these particulates and
suggests that there is a common defective mechanism inherent in the circulating cells.
Prior animal and human studies have reported that inhaled noxious pollutants
translocate from lung to systemic circulation [38], suggesting a direct link between lung to
systemic circulation. Exposure to noxious pollutants present in the smoke can possibly
contribute in excessive inflammatory response of the lungs that spills over from pulmonary
compartment and contribute in systemic inflammation [39]. Harvey et al reported that
decreased Nrf2 signalling in alveolar macrophages has been linked to altered phagocytic
activity and bacterial clearance in these cells in COPD subjects and that the Nrf2 activator
sulforaphane improve this response [40][41]. Further research is needed to explore Nrf2
signalling mechanism and role of biomass-smoke constituents in phagocytic activity of innate
immune cells.
In western population, M. catarrhalis is found in the sputum samples of S-COPD
subjects for shorter durations and is considered one of the major contributors of exacerbations
[42]. Also, P. aeruginosa has also reported in some stable S-COPD subjects and was
associated with disease severity [42]. In our study, none of the S-COPD subjects were
sampled during an exacerbation and yet colonization of M. Catarrhalis was observed.
Interestingly, in our study, we observed that BMS-COPD and H-BMS subjects harbour more
P. aeruginosa, whereas, S-COPD and HS harbour more M. catarrhalis. As reported
previously, exposure to biomass-smoke contributes to an altered bacterial community [43]. In
this study, we also observed that the increased colonization of PPBs in the airways and
impaired MDMs phagocytic activity worsened the quality of life (SQRQ and CAT) in BMS-
COPD subjects. Further investigations is required to understand the underlying mechanisms
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associated with P. aeruginosa and M. catarrhalis colonization and phagocytic activity of
macrophages.
Our study has some limitations. Firstly, this study involved identifying pathogenic
bacteria in the induced sputum samples. Upon sample collection, there is a possibility that the
induced sputum samples might be contaminated with the upper respiratory tract or oral
microbial flora. However, there is evidence to suggest that induced sputum is the most
sensitive, qualitative and quantitative method for diagnosing and infective obstructive airway
diseases [44]. Secondly, our study was gender biased i.e. H-NS, HS, and S-COPD subjects
were all males, whereas H-BMS and BMS-COPD subjects were all female. However, we did
not observe any difference in load of PPBs and phagocytic activity of MDMs among male
and female subject. It is unlikely that gender directly has any impact on macrophage
phagocytic activity, risk of developing COPD and the colonization with potentially
pathogenic bacteria. This was a real world scenario, where the female Indian population are
primarily involved in cooking and are exposed to biomass-smoke. We did make an attempt to
minimize this bias by correcting the spirometric indices for gender and performing analysis
as percentage predicted values. Also, we did not observe any gender specific difference in the
phagocytic activity of the western COPD subjects (Figure S22). Additionally, prior studies
have reported that colonization of Moraxella catarrhalis in airways is not associated with
gender [45]. Thirdly, to evaluate the phagocytic activity of MDMs, we used non-opsonized
Strep. pneumoniae and H. influenzae, which indicates that activity of opsonized receptors of
MDMs were limited in this study. Phagocytosis of opsonized bacteria is also impaired in
COPD in both alveolar macrophages and MDMs [41]. In the lung, the role of opsonisation
driving phagocytosis is limited as it is not considered a serum-rich environment.
Nevertheless, opsonisation surfactant proteins and other lung derived immunoglobulins is
possible.
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In conclusion, we have shown for the first time that subjects with non-smoking
COPD, are similar to those with S-COPD, as they harbours potentially pathogenic bacteria in
their airways that is associated with a defect in macrophage phagocytosis. This defect in
macrophages is possibly associated with increased susceptibility of those exposed to noxious
gases to develop airflow obstruction and that this is a common susceptibility factor in tobacco
smoke or biomass smoke exposure.
ACKNOWLEDGEMENTS
We would like to thank the patients who had participated in this study. We would like to
thank Mrs. Meena Raykar and Mrs. Savita Walke for their support in patient recruitment.
This study was funded by Indian Council of Medical Research (India) and Medical Research
Council (United Kingdom).
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Table 1: Demographic characteristics of the subjects
Shapiro-Wilk normality test was performed. Parametric data are expressed as mean ± SD; whereas non-parametric data are expressed as median (interquartile range). Within the group comparison was performed by Kruskal-Wallis/Dunn’s multiple comparisons test for non-parametric data and ANOVA/Tukey’s multiple comparisons test for parametric data. aP≤0.05 versus H-NS. bP≤0.05 versus HS. cP≤0.05 versus H-BMS. dP≤0.05 versus S-COPD.
[Abbreviations: COPD, chronic obstructive pulmonary disease; CAT, COPD assessment test; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; GOLD, global initiative for chronic obstructive pulmonary disease; SGRQ, St. George’s Respiratory
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Questionnaire; H-NS, healthy non-smokers; HS, smokers without COPD; H-BMS, biomass-smoke exposed healthy; S-COPD, tobacco-smoke associated COPD; BMS-COPD, biomass-smoke associated COPD, SD, standard deviation; NA, not applicable]
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Figure legends:
Figure 1: Load of potentially pathogenic microbes in the induced sputum samples
among study subjects using qPCR assay
Bacterial load of Streptococcus pneumoniae (A), Haemophilus influenzae (B), Moraxella
catarrhalis (C), and Pseudomonas aeruginosa (D) in H-NS (○ n=14), HS (☐ n=11), H-BMS
(△ n=15), S-COPD (■ n=16) and BMS-COPD (▲ n=20). The load of potentially pathogenic
bacteria was determined through specific primers (such as Spn9802 for Streptococcus
pneumoniae, P4 lipoprotein for Haemophilus influenzae, CopB for Moraxella catarrhalis,
and Pa23 for Pseudomonas aeruginosa) based qPCR SYBR Green assay. Shapiro-Wilk
normality test was performed. Data are presented as dot plots with median (interquartile
range). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed for
within the group comparisons. P<0.05 was considered statistically significant.