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Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation
Respiratory Medicine xxx (2017) 1e4
Contents lists available at ScienceDirect
Respiratory Medicine journal homepage: www.elsevier.com/locate/rmed
Short communication
Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation Dino B.A. Tan a, b, *, Jesse Armitage a, b, Teck-Hui Teo a, b, Nathanael E. Ong c, Heewoong Shin c, Yuben P. Moodley a, b, d a
Centre for Respiratory Health, School of Medicine & Pharmacology, University of Western Australia, Perth, WA, Australia Stem Cell Unit, Institute for Respiratory Health, Perth, WA, Australia School of Dentistry, University of Western Australia, Perth, WA, Australia d Department of Respiratory and Sleep Medicine, Fiona Stanley Hospital, Perth, WA, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2017 Received in revised form 6 April 2017 Accepted 25 April 2017 Available online xxx
Chronic obstructive pulmonary disease (COPD) is characterized by progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infections and increase the rates of morbidity and mortality. Currently, neither the aetiology nor pathogenesis of AECOPD are entirely understood. Exosomes have been reported to regulate immunity and inflammation via specific intercellular communications through an array of macromolecules (e.g. microRNA and proteins) contained within these microvesicles. We evaluated the level of circulating exosomes in relation to systemic inflammation in patients with AECOPD (n ¼ 20) or stable COPD (sCOPD; n ¼ 20) in comparison to non-smoking healthy controls (n ¼ 20). Exosomes in plasma were isolated by precipitation-based method, and quantified using a CD9 expression based enzyme-linked immunosorbent assay (ELISA). Plasma biomarkers of systemic inflammation, C-reactive protein (CRP), soluble tumour necrosis factor receptor-1 (sTNFR1) and interleukin (IL)-6 were also quantified using ELISA. Levels of plasma exosome were higher in AECOPD patients (p < 0.001) and sCOPD patients (p < 0.05) compared to controls. Plasma levels of CRP and sTNFR1 were highest in AECOPD, followed by sCOPD patients compared to healthy controls (p < 0.05). Plasma IL-6 was elevated in AECOPD (p < 0.05) and sCOPD patients (p < 0.01) compared to controls. The level of exosome correlated with the levels of CRP, sTNFR1 and IL-6 in plasma. Exosomes may therefore be involved in the inflammatory process of AECOPD. Further studies involving exosomal phenotyping and molecular characterization are required to fully understand their role in the pathophysiology of COPD. © 2017 Elsevier Ltd. All rights reserved.
Keywords: AECOPD COPD Exosomes Inflammation
1. Introduction Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, airway remodelling and systemic inflammation. Risk factors include cigarette smoking, air pollution and genetic predispositions [1]. A significant cause for decline in COPD are acute exacerbation (AECOPD), which is a severe sudden worsening in airway function and respiratory symptoms beyond normal daily variation, ranging from self-limited illness to critical respiratory failure requiring mechanical ventilation [1,2]. The
* Corresponding author. Level 2, MRF Building, Royal Perth Hospital, Rear 50 Murray Street, Perth, WA 6000, Australia. E-mail address: [email protected] (D.B.A. Tan).
commonest cause of AECOPD are bacterial and/or viral infections [3e5], however specific causes could not be identified in about 30% of AECOPD cases [6]. Systemic inflammatory changes in COPD and AECOPD have a strong correlation with co-morbidities such as cardiovascular disease [7,8]. In AECOPD, there is raised systemic interleukin-6 (IL-6), IL-8, reactive oxidant species and C-reactive protein (CRP) which are associated with poorer outcomes [7e11]. Understanding systemic inflammation during AECOPD will elucidate mechanisms of progression and identify new therapeutic targets that may attenuate the impact of not only airway inflammation but also the progression of co-morbidities in this condition. Circulating exosomes have been increasingly recognized as potential mediators of systemic inflammation [12,13]. Exosomes are nano-sized vesicles (40e100 nm) that are exocytosed from
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Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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List of abbreviations AECOPD BALF COPD CRP ELISA EV ExoBB GOLD HC IFNg IL miRNA mRNA PBS RNA sCOPD sTNFR1 TNFa UWA
Acute exacerbations of COPD Bronchoalveolar lavage fluid Chronic obstructive pulmonary disease C-reactive protein Enzyme-linked immunosorbent assay Extracellular vesicle Exosome binding buffer Global initiative for chronic obstructive lung disease Healthy controls Interferon-gamma Interleukin Micro ribonucleic acid Messenger ribonucleic acid Phosphate buffer saline Ribonucleic acid Stable COPD Soluble tumour necrosis factor receptor-1 Tumour necrosis factor-alpha University of Western Australia
(4 C). Clarified plasma was stored at 80 C prior to exosome isolations with the ExoQuick reagent and quantification using the CD9 ExoELISA Kit (System Biosciences, CA, USA). The exosome standards are provided in number of exosome particles as determined by the manufacturer using the Nanosight nanoparticle tracking analysis. 2.3. CD9 detection by immunogold staining and transmission electron microscopy (TEM) Exosomes were diluted 1:10 in phosphate buffered saline (PBS) and passed through a 0.22 mm centrifugal filter at 10,000g for 2 min (4 C). Exosomes were deposited onto Formvar-carbon coated grids for 20 min, blocked with 0.5% bovine serum albumin (BSA)/PBS for 10 min, incubated with mouse anti-human CD9 for 30 min and with rabbit anti-mouse IgG conjugated to 10 nm gold particles for 30 min (Sigma-Aldrich, St. Louis, MO) with PBS washes between steps. Grids were fixed then using 1% glutaraldehyde/PBS for 5 min, washed with dH2O and contrasted using 0.5% uranyl acetate for 2 min. Excess stain was blotted off and grids were dried for 2 days. Observations were carried out using a JOEL 2100 electron microscope at 120 kV (Fig. 1a and b). 2.4. Statistical analysis
multivesicular endosomes into the extracellular space and found in accessible body fluids such as blood or saliva [14,15]. These microvesicles are enriched with tetraspanin proteins (i.e. CD9, CD63 or CD81) on their surface and are key mediators of intercellular communication and contain regulatory proteins, miRNA and mRNAs [15,16]. The present study aims to investigate whether the levels of circulating exosome (CD9þ microvesicles) are abnormally elevated in individuals who experienced AECOPD and whether exosomes are associated with systemic inflammation. 2. Methods 2.1. Study subjects AECOPD patients (n ¼ 20) who were admitted to the Royal Perth Hospital (RPH) Emergency Department (Western Australia) and stable COPD patients (n ¼ 20) from a dedicated Royal Perth Hospital COPD clinic were recruited. All patients were ex-smokers (>15 pack-years and ceased smoking >1 year earlier). Healthy agematched, non-smoking subjects with no clinical evidence of COPD were also included as controls (n ¼ 20). The diagnosis and severity of COPD was categorised by a respiratory physician as per the GOLD criteria. All patients had been treated with anticholinergics, long-acting beta agonists and inhaled corticosteroids but none were receiving systemic corticosteroids or had diabetes, neuromuscular, allergic or rheumatological disease at the time of sampling. The study was approved by the Ethics Committee at the RPH (EC 2010/070) and all participants gave informed consent. 2.2. Quantification of CRP, sTNFR1, IL-6 and exosomes in plasma Plasma was separated from blood collected in lithium heparin tubes by centrifugation at 1000g for 10 min and stored at 80 C. The levels of CRP, sTNFR1 and IL-6 in plasma were quantified by ELISA (R&D Systems, MN, USA). To isolate exosomes, plasma samples were clarified by a series of centrifugation at 2000g for 10 min (4 C), followed by 10,000g for 30 min (4 C) before transferring to a 0.22 mm centrifugal filters (Sigma-Aldrich, MO, USA) and centrifuged at 10,000g for 2 min
Non-parametric Mann-Whitney U-tests were used to compare data between groups. Correlations were assessed using Spearman's rank correlation coefficient. Statistical analyses were performed with GraphPad Prism 5.04 software (La Jolla, CA, USA). 3. Results & discussion Exosomes have been studied extensively in several diseases including cancers and cardiovascular diseases [17e19], but little is known about their role in lung diseases and COPD. We reported novel data showing increased levels of circulating exosomes in AECOPD (p < 0.001) and sCOPD patients (p < 0.05) versus controls (Fig. 1c). There is a trend of elevated plasma exosomes in AECOPD versus sCOPD patients (p ¼ 0.08). Studies are describing the potentially important role of exosomes in the pathogenesis of COPD. The release of exosomes has been reported in response to cigarette smoke extract (CSE). Lung epithelial cells exposed to CSE secreted exosomes containing CCN1 (a member of the extracellular matrix-associated regulatory CCN protein family) capable of inducing the production of IL-8 [20,21], which in turn promotes the infiltration of neutrophils and may promote the development and progression of COPD [22]. Furthermore, CSE induces production of miR-210-enriched extracellular vesicles by bronchial epithelial cells that promote myofibroblast differentiation of lung fibroblasts, thus contributing to airway remodelling in COPD [23]. In other respiratory conditions, exosomes were detected in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and these exosomes induced the production of inflammatory cytokines (e.g. IFNg) by peripheral blood mononuclear cells and IL-8 by airway epithelial cells [24]. Furthermore, exosomal miRNAs isolated from BALF of patients with mild intermittent asthma resulted in a greater release of leukotrienes and IL-8 from bronchial epithelial cells [25]. Exosomes selectively deliver their contents to a target cell to regulate important processes such as cell repair, immune response and inflammation [16,26e28], and hence are likely be involved in the pathophysiology of COPD and AECOPD. The relationship between exosomes and chronic inflammation were examined by measuring levels of CRP, sTNFR1 and IL-6 in plasma. These molecules are well-established biomarkers of
Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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Fig. 1. Increased level of exosomes in patients with COPD are associated with systemic inflammation. (a) Exosomes expressing CD9 (arrows) or (b) not expressing CD9 visualized under TEM. (c) Levels of exosomes in plasma were higher in AECOPD patients than sCOPD patients and controls. The level of exosomes correlated positively with levels of (d) CRP, (e) sTNFR1 and (f) IL-6. AECOPD and sCOPD patients exhibited elevated levels of plasma (g) CRP, (h) sTNFR1 or (i) IL-6 compared to controls. ***p < 0.001; **p < 0.01; *p < 0.05. Box plots show the interquartile range (box), median (middle line) and range (whiskers) for each group.
systemic inflammation and are associated COPD-related co-morbidities (e.g. cachexia, anorexia and atherosclerosis) and mortality [7e11]. Interestingly, we demonstrated the correlation between circulating level of exosomes with plasma levels of CRP (r ¼ 0.42, p < 0.001; Fig. 1d), sTNFR1 (r ¼ 0.36, p ¼ 0.005; Fig. 1e) and IL-6 (r ¼ 0.47, p ¼ 0.006; Fig. 1f). Plasma CRP was elevated in sCOPD patients than controls (p < 0.001), and greater increase in AECOPD patients compared to sCOPD patients (p < 0.01) and controls (p < 0.001; Fig. 1g). Level of sTNFR1 was higher in AECOPD patients than in sCOPD patients (p < 0.05) and controls (p < 0.001; Fig. 1h). AECOPD and sCOPD patients exhibited higher plasma IL-6 levels than healthy controls (p < 0.05 and p < 0.01 respectively; Fig. 1i). A potential limitation of this study is that the detection of exosomes is based on the concentration of CD9 which is not expressed by all exosomes (i.e. isolated exosomes may express other tetraspanins (e.g. CD63 or CD81) but not CD9 as viewed in Fig. 1b by TEM). However, this method has been validated by the
manufacturer to highly correlate with the number of ~100 nm microparticles (i.e. exosomes) determined by the Nanosight nanoparticle tracking analysis and is calibrated to the concentration of CD9. Our study provides evidence that exosomes (i.e. CD9þ microvesicles) are elevated in COPD and AECOPD, correlate with systemic inflammatory markers and may mediate important processes during inflammation in COPD that require further investigation. Acknowledgements DT and YM conceived the project. DT and JA designed the experiments. JA, NO and HS performed the experiments and analysed the data. All authors contributed to data interpretation and manuscript preparation. DT was supported by a UWA-MRF postdoctoral fellowship and the work was funded by research grants from the Royal Perth Hospital Medical Research Foundation. The
Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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authors thank the staff at the Centre for Clinical Research in Emergency Medicine, Harry Perkins Institute of Medical Research and the COPD Linkage clinic, Royal Perth Hospital for patient recruitment, and the patients and controls who volunteered for this study. The authors also acknowledge the use of facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, University of Western Australia, a facility funded by the University, State and Commonwealth Governments. References [1] J. Vestbo, S.S. Hurd, A.G. Agustí, P.W. Jones, C. Vogelmeier, A. Anzueto, et al., Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary, Am. J. Respir. Crit. care Med. 187 (4) (2013) 347e365. [2] N. MacIntyre, Y.C. Huang, Acute exacerbations and respiratory failure in chronic obstructive pulmonary disease, Proc. Am. Thorac. Soc. 5 (4) (2008) 530e535. [3] D. Lieberman, D. Lieberman, M. Ben-Yaakov, Z. Lazarovich, S. Hoffman, B. Ohana, et al., Infectious etiologies in acute exacerbation of COPD, Diagnostic Microbiol. Infect. Dis. 40 (3) (2001) 95e102. [4] J.A. Wedzicha, Role of viruses in exacerbations of chronic obstructive pulmonary disease, Proc. Am. Thorac. Soc. 1 (2) (2004) 115e120. [5] S. Sethi, C. Wrona, K. Eschberger, P. Lobbins, X. Cai, T.F. Murphy, Inflammatory profile of new bacterial strain exacerbations of chronic obstructive pulmonary disease, Am. J. Respir. Crit. care Med. 177 (5) (2008) 491e497. [6] E. Sapey, R. Stockley, COPD exacerbations$ 2 Aetiol. Thorax 61 (3) (2006) 250e258. [7] N.J. Sinden, R.A. Stockley, Systemic inflammation and comorbidity in COPD: a result of 'overspill' of inflammatory mediators from the lungs? Review of the evidence, Thorax 65 (10) (2010) 930e936. [8] J. Miller, L.D. Edwards, A. Agusti, P. Bakke, P.M. Calverley, B. Celli, et al., Comorbidity, systemic inflammation and outcomes in the ECLIPSE cohort, Respir. Med. 107 (9) (2013) 1376e1384. [9] A. Agusti, L.D. Edwards, S.I. Rennard, W. MacNee, R. Tal-Singer, B.E. Miller, et al., Persistent systemic inflammation is associated with poor clinical outcomes in COPD: a novel phenotype, PLoS One 7 (5) (2012) e37483. [10] D. Stolz, M. Christ-Crain, N.G. Morgenthaler, J. Leuppi, D. Miedinger, R. Bingisser, et al., Copeptin, C-reactive protein, and procalcitonin as prognostic biomarkers in acute exacerbation of COPD, Chest 131 (4) (2007) 1058e1067. [11] J. Wei, X.F. Xiong, Y.H. Lin, B.X. Zheng, D.Y. Cheng, Association between serum interleukin-6 concentrations and chronic obstructive pulmonary disease: a systematic review and meta-analysis, PeerJ 3 (2015) e1199.
[12] J. Lin, J. Li, B. Huang, J. Liu, X. Chen, X.-M. Chen, et al., Exosomes: novel biomarkers for clinical diagnosis, Sci. World J. (2015) 2015. [13] F. Properzi, M. Logozzi, S. Fais, Exosomes: the future of biomarkers in medicine, Biomarkers Med. 7 (5) (2013) 769e778. [14] C. Harding, J. Heuser, P. Stahl, Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes, J. Cell Biol. 97 (2) (1983) 329e339. ry, L. Zitvogel, S. Amigorena, Exosomes: composition, biogenesis and [15] C. The function, Nat. Rev. Immunol. 2 (8) (2002) 569e579. ry, M. Ostrowski, E. Segura, Membrane vesicles as conveyors of immune [16] C. The responses, Nat. Rev. Immunol. 9 (8) (2009) 581e593. [17] G. Rabinowits, C. Gerçel-Taylor, J.M. Day, D.D. Taylor, G.H. Kloecker, Exosomal microRNA: a diagnostic marker for lung cancer, Clin. lung cancer 10 (1) (2009) 42e46. [18] Y. Kuwabara, K. Ono, T. Horie, H. Nishi, K. Nagao, M. Kinoshita, et al., Increased microRNA-1 and microRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage, Circ. Cardiovasc. Genet. 4 (4) (2011) 446e454. [19] D.D. Taylor, C. Gercel-Taylor, MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer, Gynecol. Oncol. 110 (1) (2008) 13e21. [20] H.-G. Moon, S.-H. Kim, J. Gao, T. Quan, Z. Qin, J.C. Osorio, et al., CCN1 secretion and cleavage regulate the lung epithelial cell functions after cigarette smoke, Am. J. Physiology-Lung Cell. Mol. Physiology 307 (4) (2014). L326eL37. [21] K.P. Holbourn, K.R. Acharya, B. Perbal, The CCN family of proteins: structureefunction relationships, Trends Biochem. Sci. 33 (10) (2008) 461e473. [22] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2) (2009) 215e233. [23] Y. Fujita, J. Araya, S. Ito, K. Kobayashi, N. Kosaka, Y. Yoshioka, et al., Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis, J. Extracell. Vesicles 4 (2015) 28388. [24] K.R. Qazi, P. Torregrosa Paredes, B. Dahlberg, J. Grunewald, A. Eklund, S. Gabrielsson, Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis, Thorax 65 (11) (2010) 1016e1024. [25] B. Levanen, N.R. Bhakta, P. Torregrosa Paredes, R. Barbeau, S. Hiltbrunner, J.L. Pollack, et al., Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients, J. allergy Clin. Immunol. 131 (3) (2013) 894e903. [26] T.H. Lee, E. D'Asti, N. Magnus, K. Al-Nedawi, B. Meehan, J. Rak (Eds.), Microvesicles as Mediators of Intercellular Communication in Cancerdthe Emerging Science of Cellular ‘debris’. Seminars in Immunopathology, Springer, 2011. [27] L. Luketic, J. Delanghe, P.T. Sobol, P. Yang, E. Frotten, K.L. Mossman, et al., Antigen presentation by exosomes released from peptide-pulsed dendritic cells is not suppressed by the presence of active CTL, J. Immunol. 179 (8) (2007) 5024e5032. [28] A. Beach, H.-G. Zhang, M.Z. Ratajczak, S.S. Kakar, Exosomes: an overview of biogenesis, composition and role in ovarian cancer, J. Ovarian Res. 7 (1) (2014) 1.
Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
Contents lists available at ScienceDirect
Respiratory Medicine journal homepage: www.elsevier.com/locate/rmed
Short communication
Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation Dino B.A. Tan a, b, *, Jesse Armitage a, b, Teck-Hui Teo a, b, Nathanael E. Ong c, Heewoong Shin c, Yuben P. Moodley a, b, d a
Centre for Respiratory Health, School of Medicine & Pharmacology, University of Western Australia, Perth, WA, Australia Stem Cell Unit, Institute for Respiratory Health, Perth, WA, Australia School of Dentistry, University of Western Australia, Perth, WA, Australia d Department of Respiratory and Sleep Medicine, Fiona Stanley Hospital, Perth, WA, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2017 Received in revised form 6 April 2017 Accepted 25 April 2017 Available online xxx
Chronic obstructive pulmonary disease (COPD) is characterized by progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infections and increase the rates of morbidity and mortality. Currently, neither the aetiology nor pathogenesis of AECOPD are entirely understood. Exosomes have been reported to regulate immunity and inflammation via specific intercellular communications through an array of macromolecules (e.g. microRNA and proteins) contained within these microvesicles. We evaluated the level of circulating exosomes in relation to systemic inflammation in patients with AECOPD (n ¼ 20) or stable COPD (sCOPD; n ¼ 20) in comparison to non-smoking healthy controls (n ¼ 20). Exosomes in plasma were isolated by precipitation-based method, and quantified using a CD9 expression based enzyme-linked immunosorbent assay (ELISA). Plasma biomarkers of systemic inflammation, C-reactive protein (CRP), soluble tumour necrosis factor receptor-1 (sTNFR1) and interleukin (IL)-6 were also quantified using ELISA. Levels of plasma exosome were higher in AECOPD patients (p < 0.001) and sCOPD patients (p < 0.05) compared to controls. Plasma levels of CRP and sTNFR1 were highest in AECOPD, followed by sCOPD patients compared to healthy controls (p < 0.05). Plasma IL-6 was elevated in AECOPD (p < 0.05) and sCOPD patients (p < 0.01) compared to controls. The level of exosome correlated with the levels of CRP, sTNFR1 and IL-6 in plasma. Exosomes may therefore be involved in the inflammatory process of AECOPD. Further studies involving exosomal phenotyping and molecular characterization are required to fully understand their role in the pathophysiology of COPD. © 2017 Elsevier Ltd. All rights reserved.
Keywords: AECOPD COPD Exosomes Inflammation
1. Introduction Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, airway remodelling and systemic inflammation. Risk factors include cigarette smoking, air pollution and genetic predispositions [1]. A significant cause for decline in COPD are acute exacerbation (AECOPD), which is a severe sudden worsening in airway function and respiratory symptoms beyond normal daily variation, ranging from self-limited illness to critical respiratory failure requiring mechanical ventilation [1,2]. The
* Corresponding author. Level 2, MRF Building, Royal Perth Hospital, Rear 50 Murray Street, Perth, WA 6000, Australia. E-mail address: [email protected] (D.B.A. Tan).
commonest cause of AECOPD are bacterial and/or viral infections [3e5], however specific causes could not be identified in about 30% of AECOPD cases [6]. Systemic inflammatory changes in COPD and AECOPD have a strong correlation with co-morbidities such as cardiovascular disease [7,8]. In AECOPD, there is raised systemic interleukin-6 (IL-6), IL-8, reactive oxidant species and C-reactive protein (CRP) which are associated with poorer outcomes [7e11]. Understanding systemic inflammation during AECOPD will elucidate mechanisms of progression and identify new therapeutic targets that may attenuate the impact of not only airway inflammation but also the progression of co-morbidities in this condition. Circulating exosomes have been increasingly recognized as potential mediators of systemic inflammation [12,13]. Exosomes are nano-sized vesicles (40e100 nm) that are exocytosed from
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Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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List of abbreviations AECOPD BALF COPD CRP ELISA EV ExoBB GOLD HC IFNg IL miRNA mRNA PBS RNA sCOPD sTNFR1 TNFa UWA
Acute exacerbations of COPD Bronchoalveolar lavage fluid Chronic obstructive pulmonary disease C-reactive protein Enzyme-linked immunosorbent assay Extracellular vesicle Exosome binding buffer Global initiative for chronic obstructive lung disease Healthy controls Interferon-gamma Interleukin Micro ribonucleic acid Messenger ribonucleic acid Phosphate buffer saline Ribonucleic acid Stable COPD Soluble tumour necrosis factor receptor-1 Tumour necrosis factor-alpha University of Western Australia
(4 C). Clarified plasma was stored at 80 C prior to exosome isolations with the ExoQuick reagent and quantification using the CD9 ExoELISA Kit (System Biosciences, CA, USA). The exosome standards are provided in number of exosome particles as determined by the manufacturer using the Nanosight nanoparticle tracking analysis. 2.3. CD9 detection by immunogold staining and transmission electron microscopy (TEM) Exosomes were diluted 1:10 in phosphate buffered saline (PBS) and passed through a 0.22 mm centrifugal filter at 10,000g for 2 min (4 C). Exosomes were deposited onto Formvar-carbon coated grids for 20 min, blocked with 0.5% bovine serum albumin (BSA)/PBS for 10 min, incubated with mouse anti-human CD9 for 30 min and with rabbit anti-mouse IgG conjugated to 10 nm gold particles for 30 min (Sigma-Aldrich, St. Louis, MO) with PBS washes between steps. Grids were fixed then using 1% glutaraldehyde/PBS for 5 min, washed with dH2O and contrasted using 0.5% uranyl acetate for 2 min. Excess stain was blotted off and grids were dried for 2 days. Observations were carried out using a JOEL 2100 electron microscope at 120 kV (Fig. 1a and b). 2.4. Statistical analysis
multivesicular endosomes into the extracellular space and found in accessible body fluids such as blood or saliva [14,15]. These microvesicles are enriched with tetraspanin proteins (i.e. CD9, CD63 or CD81) on their surface and are key mediators of intercellular communication and contain regulatory proteins, miRNA and mRNAs [15,16]. The present study aims to investigate whether the levels of circulating exosome (CD9þ microvesicles) are abnormally elevated in individuals who experienced AECOPD and whether exosomes are associated with systemic inflammation. 2. Methods 2.1. Study subjects AECOPD patients (n ¼ 20) who were admitted to the Royal Perth Hospital (RPH) Emergency Department (Western Australia) and stable COPD patients (n ¼ 20) from a dedicated Royal Perth Hospital COPD clinic were recruited. All patients were ex-smokers (>15 pack-years and ceased smoking >1 year earlier). Healthy agematched, non-smoking subjects with no clinical evidence of COPD were also included as controls (n ¼ 20). The diagnosis and severity of COPD was categorised by a respiratory physician as per the GOLD criteria. All patients had been treated with anticholinergics, long-acting beta agonists and inhaled corticosteroids but none were receiving systemic corticosteroids or had diabetes, neuromuscular, allergic or rheumatological disease at the time of sampling. The study was approved by the Ethics Committee at the RPH (EC 2010/070) and all participants gave informed consent. 2.2. Quantification of CRP, sTNFR1, IL-6 and exosomes in plasma Plasma was separated from blood collected in lithium heparin tubes by centrifugation at 1000g for 10 min and stored at 80 C. The levels of CRP, sTNFR1 and IL-6 in plasma were quantified by ELISA (R&D Systems, MN, USA). To isolate exosomes, plasma samples were clarified by a series of centrifugation at 2000g for 10 min (4 C), followed by 10,000g for 30 min (4 C) before transferring to a 0.22 mm centrifugal filters (Sigma-Aldrich, MO, USA) and centrifuged at 10,000g for 2 min
Non-parametric Mann-Whitney U-tests were used to compare data between groups. Correlations were assessed using Spearman's rank correlation coefficient. Statistical analyses were performed with GraphPad Prism 5.04 software (La Jolla, CA, USA). 3. Results & discussion Exosomes have been studied extensively in several diseases including cancers and cardiovascular diseases [17e19], but little is known about their role in lung diseases and COPD. We reported novel data showing increased levels of circulating exosomes in AECOPD (p < 0.001) and sCOPD patients (p < 0.05) versus controls (Fig. 1c). There is a trend of elevated plasma exosomes in AECOPD versus sCOPD patients (p ¼ 0.08). Studies are describing the potentially important role of exosomes in the pathogenesis of COPD. The release of exosomes has been reported in response to cigarette smoke extract (CSE). Lung epithelial cells exposed to CSE secreted exosomes containing CCN1 (a member of the extracellular matrix-associated regulatory CCN protein family) capable of inducing the production of IL-8 [20,21], which in turn promotes the infiltration of neutrophils and may promote the development and progression of COPD [22]. Furthermore, CSE induces production of miR-210-enriched extracellular vesicles by bronchial epithelial cells that promote myofibroblast differentiation of lung fibroblasts, thus contributing to airway remodelling in COPD [23]. In other respiratory conditions, exosomes were detected in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and these exosomes induced the production of inflammatory cytokines (e.g. IFNg) by peripheral blood mononuclear cells and IL-8 by airway epithelial cells [24]. Furthermore, exosomal miRNAs isolated from BALF of patients with mild intermittent asthma resulted in a greater release of leukotrienes and IL-8 from bronchial epithelial cells [25]. Exosomes selectively deliver their contents to a target cell to regulate important processes such as cell repair, immune response and inflammation [16,26e28], and hence are likely be involved in the pathophysiology of COPD and AECOPD. The relationship between exosomes and chronic inflammation were examined by measuring levels of CRP, sTNFR1 and IL-6 in plasma. These molecules are well-established biomarkers of
Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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Fig. 1. Increased level of exosomes in patients with COPD are associated with systemic inflammation. (a) Exosomes expressing CD9 (arrows) or (b) not expressing CD9 visualized under TEM. (c) Levels of exosomes in plasma were higher in AECOPD patients than sCOPD patients and controls. The level of exosomes correlated positively with levels of (d) CRP, (e) sTNFR1 and (f) IL-6. AECOPD and sCOPD patients exhibited elevated levels of plasma (g) CRP, (h) sTNFR1 or (i) IL-6 compared to controls. ***p < 0.001; **p < 0.01; *p < 0.05. Box plots show the interquartile range (box), median (middle line) and range (whiskers) for each group.
systemic inflammation and are associated COPD-related co-morbidities (e.g. cachexia, anorexia and atherosclerosis) and mortality [7e11]. Interestingly, we demonstrated the correlation between circulating level of exosomes with plasma levels of CRP (r ¼ 0.42, p < 0.001; Fig. 1d), sTNFR1 (r ¼ 0.36, p ¼ 0.005; Fig. 1e) and IL-6 (r ¼ 0.47, p ¼ 0.006; Fig. 1f). Plasma CRP was elevated in sCOPD patients than controls (p < 0.001), and greater increase in AECOPD patients compared to sCOPD patients (p < 0.01) and controls (p < 0.001; Fig. 1g). Level of sTNFR1 was higher in AECOPD patients than in sCOPD patients (p < 0.05) and controls (p < 0.001; Fig. 1h). AECOPD and sCOPD patients exhibited higher plasma IL-6 levels than healthy controls (p < 0.05 and p < 0.01 respectively; Fig. 1i). A potential limitation of this study is that the detection of exosomes is based on the concentration of CD9 which is not expressed by all exosomes (i.e. isolated exosomes may express other tetraspanins (e.g. CD63 or CD81) but not CD9 as viewed in Fig. 1b by TEM). However, this method has been validated by the
manufacturer to highly correlate with the number of ~100 nm microparticles (i.e. exosomes) determined by the Nanosight nanoparticle tracking analysis and is calibrated to the concentration of CD9. Our study provides evidence that exosomes (i.e. CD9þ microvesicles) are elevated in COPD and AECOPD, correlate with systemic inflammatory markers and may mediate important processes during inflammation in COPD that require further investigation. Acknowledgements DT and YM conceived the project. DT and JA designed the experiments. JA, NO and HS performed the experiments and analysed the data. All authors contributed to data interpretation and manuscript preparation. DT was supported by a UWA-MRF postdoctoral fellowship and the work was funded by research grants from the Royal Perth Hospital Medical Research Foundation. The
Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014
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Please cite this article in press as: D.B.A. Tan, et al., Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation, Respiratory Medicine (2017), http://dx.doi.org/10.1016/j.rmed.2017.04.014