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We thank Dr Zhang and colleagues for their comments on our paper1. We certainly agree that in this emerging field of extracellular vesicle (EV) research, it is vital that identification and characterisation of different EV populations are as robust as possible. To this end, we very much welcome detailed discussions on methodologies used for each study, to enhance and improve the quality of EV-related work within the lung research community.
In our paper, we specifically chose to examine the role of microvesicles (MVs) in acute lung injury (ALI), and the roles of apoptotic bodies and exosomes are beyond the scope of the study. We do not exclude the presence of apoptotic bodies or surfactant micelles in our in vivo samples, or indeed single or clustered MVs larger than 1µm, however our surface marker analysis of MV subpopulations by flow cytometry was deliberately conservative and limited to only events below the conventional size cut off of 1µm. Hence figure 3 of our paper shows effectively only one EV population, i.e. MVs. For our isolation of MVs for functional studies, we used differential centrifugation to enrich MVs but these technical matters were discussed in some detail in the published manuscript.
Dr Zhang and colleagues have concerns about the dose of LPS (20µg) used in our in vivo ALI model. However, intratracheal (i.t.) instillation of high dose LPS (20µg or more per mouse) is a clinically-relevant, well established model of AL...
Dr Zhang and colleagues have concerns about the dose of LPS (20µg) used in our in vivo ALI model. However, intratracheal (i.t.) instillation of high dose LPS (20µg or more per mouse) is a clinically-relevant, well established model of ALI, used very widely by investigators in ALI research including ourselves2-5. Dr Zhang stated that large doses of LPS often result in release of apoptotic bodies but few MVs from alveolar macrophages, but we wonder if this statement is based on in vitro experiments using non-primary cells, rather than in vivo ALI models? Dr Zhang’s group recently showed6 the production of apoptotic bodies with 1µg LPS treatment, but their results were obtained using an immortalised cell line (MH-S alveolar macrophages) in vitro, rather than primary alveolar macrophages in vivo. Interestingly, they observed that apoptotic body production peaked later (at 6 hours) when primary cells (bone marrow derived macrophages) were treated with LPS in vitro, highlighting a clear difference between primary cells and immortalised cell lines (such as RAW cells, THP-1 and MH-S cells)6. While we cannot entirely exclude the possibility that some apoptotic bodies were produced within our model, it has been shown that i.t. LPS in vivo does not initiate apoptosis of alveolar macrophages until much later time points7,8. Taken together, we believe that concerns regarding apoptotic bodies influencing our conclusions are unsubstantiated for the acute responses investigated in our model. This is of course not to say that the release of apoptotic bodies or other EVs does not play an important role during subsequent phases of ALI pathophysiology.
Sanooj Soni, Michael R Wilson, Kieran P O’Dea, Masao Takata
Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, UK
1. Soni S, Wilson MR, O'dea KP, et al. Alveolar macrophage-derived microvesicles mediate acute lung injury. Thorax 2016;71(11):1020-29.
2. Woods SJ, Waite AA, O'Dea KP, et al. Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation. American Journal of Physiology-Lung Cellular and Molecular Physiology 2015;308(9):L912-L21.
3. Gong J, Wu Zy, Qi H, et al. Maresin 1 mitigates LPS‐induced acute lung injury in mice. British journal of pharmacology 2014;171(14):3539-50.
4. Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nature medicine 2012;18(5):759-65.
5. Dorr AD, Wilson MR, Wakabayashi K, et al. Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies. Journal of Applied Physiology 2011;111(1):177-84.
6. Zhu Z, Zhang D, Lee H, et al. Macrophage-derived apoptotic bodies promote the proliferation of the recipient cells via shuttling microRNA-221/222. Journal of Leukocyte Biology 2017:jlb. 3A1116-483R.
7. Vernooy JH, Dentener MA, Van Suylen RJ, et al. Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: no role for tumor necrosis factor-α and infiltrating neutrophils. American journal of respiratory cell and molecular biology 2001;24(5):569-76.
8. Kearns MT, Barthel L, Bednarek JM, et al. Fas ligand-expressing lymphocytes enhance alveolar macrophage apoptosis in the resolution of acute pulmonary inflammation. American Journal of Physiology-Lung Cellular and Molecular Physiology 2014;307(1):L62-L70.
We are writing to comment on the work entitled “Alveolar macrophage-derived microvesicles mediate acute lung injury” published by Dr. Soni et al on Thorax 2016; 71:1020-1029.
Our group focuses on lung extracellular vesicle (EV) research and also studied the inhaled LPS-induced EVs in mouse models. Based on our experience, we raise the following comments to the work done by Dr. Soni et al and wish to draw attentions to future EV researchers. EV research is a novel field and carries a promising potential for the development of diagnostic and therapeutic agents. However, given the early stage of EV research, particular in the field of lung injury, the consistency of results relies largely on the precise techniques used in the isolation and characterization of these vesicles.
Briefly, EV is currently classified into three major categories per the definition of Society of extracellular vesicle research . Apoptotic bodies (ABs) are the largest sizes of EVs usually larger than 1 µm and often resulted from cell death. Microvesicles (MVs) are the middle sized EVs (200 nm-1 µm) and are generated via plasma membrane budding. Exosomes (Exos) are the smallest EVs (less than 200 nm) and often generated from IVB-ER-Golgi system. Due to the different mechanisms of generation, MVs and Exos usually favor different compositions and subsequently may carry differential downstream biological functions[3 4]. For example, Exos have been reported to carry t...
Briefly, EV is currently classified into three major categories per the definition of Society of extracellular vesicle research . Apoptotic bodies (ABs) are the largest sizes of EVs usually larger than 1 µm and often resulted from cell death. Microvesicles (MVs) are the middle sized EVs (200 nm-1 µm) and are generated via plasma membrane budding. Exosomes (Exos) are the smallest EVs (less than 200 nm) and often generated from IVB-ER-Golgi system. Due to the different mechanisms of generation, MVs and Exos usually favor different compositions and subsequently may carry differential downstream biological functions[3 4]. For example, Exos have been reported to carry the very minimal amount of popular microRNAs (miRNAs). Even for those most promising miRNA markers, less than 1 copy of miRNA can be found in each Exo. This is part of the reason why the classification of EVs may be important.
In the work presented by Dr. Soni et al, a very large dose of LPS (20 μg per mouse) has been delivered to mice via intratracheal instillation. Certainly, different batch of LPS may carry different levels of potency. However, we found that the larger dose of LPS often quickly resulted in the release of ABs but rather than MVs by alveolar macrophages. In our experiences, as little as 1 μg LPS (Sigma-Aldrich, cat #L2630, St. Louis, MO, USA) resulted in the robust amount of MVs (about 50% in total EVs). However, a fair amount of Exos (about 25% in total EVs) is still mixed in the LPS-induced EVs. Additionally, the FACS analysis presented in figure 3 shows only one population of vesicles. This could be due to the overly too large dose of LPS and the detected EVs fall mainly on the range of ABs. In our experience, using a smaller dose of LPS, we often clearly observe two different population of EVs, one belongs to the larger size of ABs, and the other belongs to the smaller MVs.
We recognize that the variability of results could result from different technique, detecting apparatus, different batch of LPS and also different size/age/strain of mice. However, we would like to discuss our findings on this emerging novel topic and our comments may be found interesting and useful by other audience.
Thank you for your time and attention!
1. Soni S, Wilson MR, O'Dea KP, et al. Alveolar macrophage-derived microvesicles mediate acute lung injury. Thorax 2016;71(11):1020-29. doi: 10.1136/thoraxjnl-2015-208032
2. Gyorgy B, Szabo TG, Pasztoi M, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011;68(16):2667-88. doi: 10.1007/s00018-011-0689-3
3. Yanez-Mo M, Siljander PRM, Andreu Z, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 2015;4 doi: ARTN 27066
4. Raposo G, Stoorvogel W. Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol 2013;200(4):373-83. doi: 10.1083/jcb.201211138
5. Alexander M, Hu RZ, Runtsch MC, et al. Exosome-delivered microRNAs modulate the inflammatory response to endotoxin. Nat Commun 2015;6 doi: ARTN 7321