What is the role of microvesicles in the development of acute lung injury?

Lead Research Organisation: Imperial College London
Department Name: Dept of Surgery and Cancer

Abstract

Scientific Context
Acute lung injury (ALI) is a life-threatening lung condition, frequently diagnosed in critical unwell patients. Unfortunately, sufferers often require long stays in intensive care wards, at a major cost to the NHS and currently no drug treatment exists. Patients with ALI have 40% chance of dying and those that survive have severe long-term physical and psychological problems. Therefore urgent research is needed to improve scientific knowledge of this lethal condition and identify new therapies.

Acute Lung Injury
In this condition, there is a sudden failure of the breathing system, with difficulty getting oxygen into the lungs. Therefore oxygen cannot get into the blood and cannot be delivered to other organs. It is a lethal condition, affecting both children and adults, and kills 40% of patients who develop the condition. It usually affects those patients who are already very unwell due to another disease or who have major injuries.

Research question and Importance
Microvesicles (MVs) are very small particles released by cells when they are damaged, injured or inflamed. Recent research has demonstrated that MVs carry messages between cells and it is now known that they have an important role in cell-to-cell communication. As such, MVs have been shown to have important roles in many inflammatory conditions such as diabetes, coronary artery disease and hypertension. However their role has not been examined in ALI, which is another inflammatory disease. My initial results have found that MVs are present in ALI and they carry important inflammatory molecules. We have also found that they can cause inflammatory reactions in previously, healthy cells. We will examine whether MVs have a major role in ALI. If our results demonstrate that MVs play a major role in ALI, this would allow development of new medicines that can stop MVs and thus treat this deadly disease.

Research Methods
I am performing this project under the direction and guidance of my supervisors (Professor Takata, Dr Michael Wilson, Dr Kieran O'Dea) at the Section of Anaesthetics, Pain Medicine and Intensive Care at Imperial College London. Part of this study will also take place in National Heart and Lung Institute at Imperial College with Professor Adcock and Dr Mark Perry. The project will involve detailed laboratory experiments working with cells found in the lungs. MVs will be created from these lung cells and we will identify their exact function and whether they can directly activate other cells found in the lungs. We will also investigate whether inhibiting the production or absorption of MVs in the lungs, will ease symptoms and signs of ALI. Furthermore, we will explore and identify what messages these MVs carry between lung cells.

Technical Summary

Aims and Objectives
I aim to establish the significance of Microvesicles (MVs) in Acute Lung Injury (ALI), investigating three central objectives:
1. To explore if patterns of MV release can be used to distinguish between the phases of ALI.
2. To investigate the inflammatory mediators and biological cargo carried in MVs
3. To identify the role of MVs in the pathogenesis, potentially inspiring novel therapeutic targets.

Methodology
I will use clinically relevant mouse models of ALI, where the acute inflammatory, fibrotic and resolution phases are readily identifiable. MV kinetics/profiles (i.e. number and cell origin) will be identified in these phases by flow cytometry and will be compared. To characterise the information relayed through MV in ALI, MV pellets will be collected from different acute and chronic ALI models and intra-vesicular mediators (e.g. interleukins and cytokines) will be measured by ELISA/flow cytometry. RNA from MVs will be extracted and quantified through real-time RT-PCR. To characterise the functional significance of MVs in ALI, I will produce MVs from alveolar cells and incubate them with other alveolar cells both in-vitro and in-vivo. Cellular responses will be assessed to determine if MVs can activate intra-alveolar cells. Attenuation of these responses in models where MV production/uptake is inhibited (i.e. in knock-out mice or though pharmacological means) will also be assessed.

Scientific and medical opportunities
These results will provide crucial information for the scientific community on the pathophysiology of ALI, potentially identifying a novel, previously unexplored communication pathway between intra-alveolar cells. This may explain why therapies for ALI to date have proved ineffective and as such the results from this project may allow the development of new opportunities for therapeutic strategies for ALI.

Planned Impact

Our aim, in this basic science/translational study, is to investigate the biological and functional role of microvesicles (MVs) in acute lung injury (ALI). Due to the lack of effective therapies for ALI, there remains an urgent need for a redirection in ALI research to identify new treatments. MVs may provide valuable information about the pathophysiological process of ALI and may prove to be novel therapeutic targets. Consequently, this project may have a significant impact amongst academics, clinicians, patients, the NHS and potentially, commercial private sectors.

Academic Impact
There are over 21000 journal articles on ALI yet despite this intense research activity, academic understanding of ALI contains large discrepancies, mortality remains unacceptably high (~40%). ALI management is hampered by the lack of any pharmaceutical treatment, diagnostic tool or biomarkers. MVs have the potential to fulfill all three roles. MVs have already been implicated in the pathophysiology and as potential biomarkers in a number of other inflammatory conditions. However there is a paucity of literature regarding the role of MVs in ALI. Due to the diverse and clinically relevant ALI models developed in our laboratory, we are in a unique position to investigate the role of MVs in ALI. Therefore this project has an excellent opportunity to enhance the knowledge and expertise of ALI for academics, identify and explore a previously unknown mechanism of intra-alveolar cell communication and potentially highlight a new therapeutic target. We have developed protocols that readily identify which cells MVs are derived from (see case for support for data), this research could have significant impact in academia as MVs may resolve several controversies in ALI e.g. the source of inflammatory mediators in the intra-alveolar space. We will continue to develop our novel and innovative methodology, so that our techniques can impact and inspire other laboratories to measure MVs in other inflammatory conditions.

Clinical Impact
Our preliminary results show that MVs are readily measureable in bronchoalveolar lavage fluid from an ALI model and that these MVs are carriers of biological cargo in ALI including TNF alpha. Furthermore alveolar macrophage-derived MVs act as vectors, transferring RNA between intra-alveolar cells. Our pilot data also illustrates that MVs are functionally active and can activate endothelial cells in-vitro. Consequently MVs may be heavily implicated in the pathophysiology of ALI and although this project is basic science in concept, it may provide major steps in the establishment of pharmaceutical therapies for ALI. Furthermore, MVs may be a valuable diagnostic and disease-monitoring tool as they are readily measured in bronchoalveolar lavage fluid. As such, MVs may have a significant clinical impact in the management of ALI.

Business Impact
Due to the novelty of this research and it's potential impact on clinicians, academics and patients, the project may attract grants/research and development funds from businesses/pharmaceutical industry.

Society Impact
The mortality from ALI remains unacceptably high (~40%). Additionally those patients that survive have significant physical and psychological morbidity. These unfavorable statistics are certainly related to complex, pathogenesis of this syndrome with multiple signaling pathways activated. Our research may increase the knowledge surrounding the pathophysiology of ALI and provide major steps in the establishment of pharmaceutical therapies for ALI. These would improve mortality figures, enhancing the quality life, health and well-being our patients.

Publications

10 25 50
 
Description Surgical cancellation rates due to peri-operative hypertension: Implementation of multi-disciplinary guidelines across primary and secondary care.
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact Patients with uncontrolled hypertension are at increased risk of complications during general anaesthesia but the number of patients whose surgery is delayed/cancelled due to hypertension remains unknown. Prospective, regional multicentre service evaluations were performed on consecutive patients undergoing elective surgery before (October 2013 (n=7673)) and after publication of 'The management of hypertension before elective surgery' guidelines (Association of Anaesthetist's of Great Britain and Ireland (AAGBI)/British Hypertensive Society (BHS)) (November 2018 (n=7100)) to quantify the number of operations cancelled due to hypertension alone and assess impact of the guidelines on cancellation rates. In October 2013, 1.37% (105/7673) of patients listed for elective surgery were cancelled solely due to raised blood pressure but this significantly reduced to 0.54% (38/7100, p<0.001) in 2018. There has been a significant reduction in the number of cancellations for stage 1 or 2 hypertension (2013: 72 vs 2018: 14, p<0.001) in keeping with the recommendations in the guidelines. Furthermore, the number of patients being referred back to primary care for the management of hypertension has reduced (2013: 82 vs 2018: 30, p<0.001). Our data suggests achievement of three major outcomes: 1) Reduced surgical cancellations due to hypertension alone; 2) Improved detection of significant hypertension before elective surgery; 3) Reduced referral back to primary care from hospital for hypertension management. To the best of our knowledge, this is the first time the successful implementation of guidelines has been assessed on such a broad scale. Our data demonstrates the overwhelming integration of these guidelines in both primary and secondary care which is likely to have made a positive psychosocial, physical and economic impact to patients and the NHS.
 
Description member of the PLAATAG committee
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
 
Description ERS Research Fellowship
Amount € 2,500 (EUR)
Organisation European Respiratory Society (ERS) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2020 
End 09/2020
 
Title ATP selectively packages TNF within microvesicles released from alveolar macrophages 
Description ATP triggers cells to release microvesicles (MVs), which play crucial roles in cellular communication by ferrying molecular cargo between cells. MVs may protect its contents from dilution, degradation or consumption, yet there is a paucity of literature examining TNF release within intra-alveolar MVs. We aimed to investigate if ATP causes alveolar macrophages (AM) to preferentially release TNF within MVs. Primary mouse AMs were activated with 1µg/ml LPS, and then exposed to a 'second hit' of ATP 6mM/ecto-ATPase inhibitor 1mM for 1 hour to produce MVs (identified on flow cytometry as CD11c+, <1µm, detergent-sensitive events). TNF/IL-6 content of MV pellets and MV-free supernatants (separated by high-speed centrifugation) were analyzed by ELISA/western blotting and compared to control (LPS stimulation alone). 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact ATP treatment eradicated soluble TNF release from AMs (Control 107±13 pg vs ATP 4.8±6.1 pg, p<0.01 N=5-7) but substantively upregulated TNF content within MVs (0.5±3.6 vs 26.8±8.5, p<0.01 N=5-7) as measured by ELISA. Western blotting confirmed these findings including 17kDa TNF within MVs. ATP did not significantly alter the profile of IL-6 release in the soluble (Control 14.53±6.0 pg vs ATP 6.0±0.5 pg, N=3-4) or MV fraction (3.1±0.7 vs 2.4±0.3 pg, N=3-4), suggesting this 'switch' may be specific to TNF. These data show that a danger signal such as ATP, superimposed onto a pro-inflammatory stimulus, suppresses TNF release from AMs in the ordinary soluble form but preferentially packages TNF within MVs. This switch phenomenon indicates a novel mechanism of long-range TNF signalling in inflammatory lung diseases such as acute lung injury. 
 
Title Alveolar macrophages preferentially take up microvesicles in the alveolar space 
Description Background: Microvesicles (MV) are important mediators of inter-cellular communication within the alveolar space, causing significant alveolar epithelial cell (AEC) injury (Soni et al. Thorax 2016;71:1020-29). Here we investigate, for the first time, MV uptake profile by individual intra-alveolar cells during resting/inflamed lung conditions. Methods: Macrophage- or murine lung epithelial cell (MLE)-derived MVs were labelled with membrane intercalating dye DiD, and incubated for 1-4h with coculture of primary alveolar macrophages (AM) and MLE cells, with/without LPS priming. AM-derived or in vivo-generated MVs (a mixed population consisting mainly of AM/AEC-derived MVs harvested from intratracheal (i.t.) LPS treated mice) were DiD-labelled and i.t. instilled into mice for 1h, with/without LPS pre-treatment. Cellular MV uptake was evaluated by flow cytometry, using cell surface markers and DiD mean fluorescence intensity (MFI). Results: In vitro, the majority of MVs were taken up by AMs compared to MLE cells, irrespective of MV cell sources or LPS priming. In vivo, preferential uptake by AMs vs AEC was also observed: for AM-derived MVs, AM MFI 1175±544 vs AEC MFI 8±2, p<0.05 n= 3; for in vivo-derived MVs, AM MFI 1598±166 vs AEC MFI 15±2, p<0.001 n=3. In LPS-pretreated mice, this pattern did not change. Conclusion: Surprisingly, and in contrast to a previous report of significant MV uptake by AEC (Bourdonnay et al. J Exp Med 2015;212:729-42), we found that MV uptake/internalisation was performed almost exclusively by AMs, irrespective of MV phenotype under both resting and inflamed conditions. The results strongly suggest that AMs play a crucial modulating role in MV-mediated intra-alveolar inflammation. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact Background: Microvesicles (MV) are important mediators of inter-cellular communication within the alveolar space, causing significant alveolar epithelial cell (AEC) injury (Soni et al. Thorax 2016;71:1020-29). Here we investigate, for the first time, MV uptake profile by individual intra-alveolar cells during resting/inflamed lung conditions. Methods: Macrophage- or murine lung epithelial cell (MLE)-derived MVs were labelled with membrane intercalating dye DiD, and incubated for 1-4h with coculture of primary alveolar macrophages (AM) and MLE cells, with/without LPS priming. AM-derived or in vivo-generated MVs (a mixed population consisting mainly of AM/AEC-derived MVs harvested from intratracheal (i.t.) LPS treated mice) were DiD-labelled and i.t. instilled into mice for 1h, with/without LPS pre-treatment. Cellular MV uptake was evaluated by flow cytometry, using cell surface markers and DiD mean fluorescence intensity (MFI). Results: In vitro, the majority of MVs were taken up by AMs compared to MLE cells, irrespective of MV cell sources or LPS priming. In vivo, preferential uptake by AMs vs AEC was also observed: for AM-derived MVs, AM MFI 1175±544 vs AEC MFI 8±2, p<0.05 n= 3; for in vivo-derived MVs, AM MFI 1598±166 vs AEC MFI 15±2, p<0.001 n=3. In LPS-pretreated mice, this pattern did not change. Conclusion: Surprisingly, and in contrast to a previous report of significant MV uptake by AEC (Bourdonnay et al. J Exp Med 2015;212:729-42), we found that MV uptake/internalisation was performed almost exclusively by AMs, irrespective of MV phenotype under both resting and inflamed conditions. The results strongly suggest that AMs play a crucial modulating role in MV-mediated intra-alveolar inflammation. 
 
Title Confocal Microscopy of microvesicles 
Description CONFOCAL MICROSCOPY - STAINING PROTOCOL Preparation: Fix cells with paraformaldehyde Ideally use: 6-well plates 24 x 40mm coverslips Use antibody manufacturer fluorescent staining for immunohistochemistry protocol/datasheet Have 3+ controls for antibody dose response on first use Staining protocol: Wash wells with PBS x1 Permeabilise with 0.5% Triton Leave for 3mins Wash with PBS Add 3% BSA to cover wells Leave for 30mins (covered with foil on rotator) Add 1.5% BSA to cover wells Place primary blocking antibody on to labeled parafilm sheet Use 3 different concentrations (e.g. on control wells) for serial antibody dilution Have negative control with no blocking antibody Put coverslips (with cells facing down) onto parafilm sheet/antibody Leave for recommended time (as per manufacturer datasheet) Place coverslips back in well plates (turn coverslips over) Wash with PBS x3 Leave for 5mins after each wash (covered in dark) Add secondary antibody to all wells e.g. 2% donkey serum (2mg/ml), 1 in 1000, diluted in PBS Add 100ul of antibody to each well Leave for 1 hour Wash with PBS x3 Leave for 5mins after each wash Add DAPI intra-nuclear stain 1 in 10000 solution (pre-made); add enough to cover coverslip Leave for 10mins Place coverslips on separate labeled slides (with cells facing down) Add Mounting PermaFluor solution to slides before placing coverslips Leave for 30mins View under confocal/fluorescent microscope 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact Able to visualise microvesicles and the cargo their carry with confocal microscopy 
 
Title Electron Microscopy of microvesicles 
Description Immune EM/Transmission EM Stimulate cells in 35x15mm plates in 500µl of stimulant to produce MVs Following stimulation wash with fitered PBS twice to remove any remnant stimulatory factors Then add 2%PFA in cocdylate buffer for 1hr (bring samples to harefiled) Once arrive, wash three times with filtered cocoylate PBS (leave in or 2 minute) Then add TNF (1 in 40 made) up in 1.5%BSA and cocdylate buffer (500µL in originial dish - leave overnight) Next morning wash three time in codylate buffer Then add 1:50 gold antibody - leave for 1 hr Wash three time with codoylate buffer Then add 2.5%glyteraldehyde for 1hr Wash three times again with cacodylate Then put in osmium tetroxide 1% for 1 hr Then wash twice with cocdylate Then add 1. Cover with ethanol 25% for 20 minutes. 2. Discard ethanol 25% and add ethanol 50% for 20 minutes. 3. Discard ethanol 50% and add ethanol 70% for 20 minutes. 4. Discard ethanol 70% and add ethanol 95% for 20 minutes. 5. Discard ethanol 95 % and add ethanol 100% for 20 minutes (x2). 6. Discard ethanol 100% and add propylene oxide rotating the dish until a layer appears. Transfer the layer to an eppendorf and spin. 7. Remove supernatant and add araldite propylene 1:1. Leave on rotator overnight. Next day: 1. Discard the araldite/propylene and add new araldite. . 2. Then put the vials on the rotator for at least 2 hours. 3. Discard the araldite and add new araldite again. 4. Then put the vials on the rotator for at least 2 hours. 5. Put sample label into embedding capsule. 6. Fill with araldite. 7. Put the samples in the proper capsule. 8. Polymerise in oven over 60 o C for 48 hrs. Ultra-thin sections (100 nm) were cut using a Diatome diamond knife, floated onto distilled water, collected on grids and stained with 2% uranyl acetate and lead citrate for 10 minutes in each solution. For the EM evaluation, we acquired images using the Transmission Electronic Microscope (TEM) JEOL 1200 EX, and digital images were captured using GatanDigitalMicrograph. SEM: protocol 1. Label properly the petri dishes before the use. (after stimulation) 2. Culture cells in a Petri dish and add stimulator according to protocol. 3. Remove the medium and add glutaraldehyde 2.5% for 1 hour. 4. Remove glutaraldehyde and wash with cacodylate buffer. 5. Put in osmium tetroxide 1% for 1 hr. 6. Wash again with cacodylate buffer. 7. Cover with tannic acid 1% for 20 minutes. 8. Remove tannic acid and add ethanol 25% for 20 minutes. 9. Discard ethanol 25% and add ethanol 50% for 20 minutes. 10. Discard ethanol 50% and add ethanol 70% for 20 minutes. 11. Discard ethanol 70% and add ethanol 95% for 20 minutes. 12. Discard ethanol 95 % and add ethanol 100% for 20 minutes (x2). 13. Discard ethanol 100%. 14. Air dry using HDMS hexamethyldisilazane. 15. Fixed onto aluminum stub with AU/PA. 8. Read using SEM micros 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact we have been able to visualise microvesicles budding off from cells as well as some of the cargo they carry. 
 
Title Macrophages preferentially package TNF within microvesicles in response to ATP 
Description TNF is a potent pro-inflammatory cytokine, playing a crucial role in various inflammatory diseases. Microvesicles (MVs), carrying a variety of mediators including cytokines, provide a vital alternative pathway for inflammatory signalling, yet there is a paucity of literature examining MV-associated TNF transfer. We investigated the effect of ATP, a typical danger signal that also stimulates MV production, on the manner of TNF release by macrophages. RAW 264.7 cells were stimulated with 1µg/ml LPS for 1 hour, and then treated with an additional 'second hit' of ATP 6mM/ecto-ATPase inhibitor 1mM for 1 hour to produce MVs. Cells were analysed by flow cytometry for TNF expression. Cell-free supernatant underwent high speed centrifugation to separate MV pellet and MV-free supernatant, and TNF/IL-6 content of both fractions were quantified by ELISA/western blotting. Controls included cells stimulated with LPS alone for 2 hours. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Compared to LPS alone, surface TNF expression on RAW cells was lower with the second hit of ATP (20±3 vs 2±2, mean fluorescent intensity, p<0.01 N=3). ATP treatment almost abolished soluble TNF release into the media (471±127 vs 32±14 pg, p<0.05 N=3) but substantively upregulated TNF content within MV (1.1±0.2 vs 23±7 pg, p<0.05 N=3), as measured by ELISA. Western blotting confirmed the presence of TNF within MVs. The profile of IL-6 release between the soluble vs MV-associated fractions were not altered by ATP, suggesting this 'switch' phenomenon may be specific to TNF. These data show, for the first time, that ATP preferentially packages TNF within MVs, whilst concurrently inhibiting soluble TNF release. Our results indicate that danger signals superimposed onto pro-inflammatory signals can switch macrophage TNF release from the ordinary soluble from to the MV-associated form, suggesting a novel mechanism of long-range MV-associated TNF transfer in case of significant tissue injury. 
 
Title Microvesicles Mediate Long-range Membrane Tnf Signalling, Inducing Inflammatory Changes Consistent With Acute Lung Injury 
Description Rationale TNF plays a key role in inflammatory lung diseases including acute lung injury (ALI). We previously demonstrated the presence of TNF within microvesicles (MVs) released in the alveoli during a LPS-induced model of ALI. Although MVs form an ideal vehicle for ferrying 'non-classically secreted' cytokines (e.g. Il-1ß/IL-18), it is unknown why TNF, a 'classically secreted' cytokine, is released within MVs. Here we investigate the hypothesis that MV-mediated TNF secretion allows protected, more efficient TNF signalling compared to its soluble counterpart. Methods Bone marrow derived macrophages (BMDMs) were harvested from C57/BL6 mice, stimulated with LPS and then ATP to generate TNF-laden MVs. MVs and their TNF cargo were characterised by western blotting, confocal and immune-electron microscopy. These TNF+ve MVs were then instilled into the lungs of untreated mice, and after 4 hours their biological effects compared to BMDM-derived MVs harvested from TNF-/- mice (TNF-ve MVs). Recombinant TNF (100ng/ml, 50µL) was also instilled to compare the effects of MV-mediated vs soluble TNF signalling. Results Intriguingly, BMDMderived MVs carried the pro-TNF (26kDa) isoform rather than the soluble (17kDa) isoform, as assessed by western blotting. Immuneelectron/ confocal microscopy demonstrated that macrophages actively packaged pro-TNF within MVs during formation, which eventually localised to vesicle membranes. TNF+ve MVs induced significant intra-alveolar inflammation compared to TNF-ve MVs: lung inflammatory monocyte infiltration (410,700 ± 38,700 vs 214,000 ± 28,800 cells/ml, p<0.01, n=4); BALF protein (0.34 ± 0.03 vs 0.24 ± 0.01 mg/ml, p<0.05, n=4); ICAM-1 expression on epithelial cells (mean fluorescence intensity: 317 ± 36 vs 214 ± 14, p<0.05 n=4); and BALF KC levels (2746 ± 285 vs 1854 ± 134 pg/ml, p<0.05 n=4). Unlike the potent effects of MV-laden TNF, intratracheal soluble TNF did not produce significant inflammation, despite its dose exceeding the amount of proTNF in the MVs. Conclusion Our data demonstrate, for the first time, that danger signals (e.g. ATP) secrete TNF as the membrane pro-TNF isoform within MVs, inducing intra-alveolar inflammation (consistent with ALI) in a TNF-dependent manner. The soluble form of TNF (normally released within alveoli) could be quickly degraded/neutralised and would not reach target cells very effectively. In contrast, pro-TNF enclosed within MVs seems to be protected, allowing efficient longer-range signalling. Furthermore, we provide an alternative hypothesis for membrane TNF signalling in inflammatory lung disease (previously thought to occur only upon direct cell-to-cell contact): membrane TNF can be secreted within shed MVs, activating more-distant target cells without cell contact per se. : 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? No  
Impact Rationale TNF plays a key role in inflammatory lung diseases including acute lung injury (ALI). We previously demonstrated the presence of TNF within microvesicles (MVs) released in the alveoli during a LPS-induced model of ALI. Although MVs form an ideal vehicle for ferrying 'non-classically secreted' cytokines (e.g. Il-1ß/IL-18), it is unknown why TNF, a 'classically secreted' cytokine, is released within MVs. Here we investigate the hypothesis that MV-mediated TNF secretion allows protected, more efficient TNF signalling compared to its soluble counterpart. Methods Bone marrow derived macrophages (BMDMs) were harvested from C57/BL6 mice, stimulated with LPS and then ATP to generate TNF-laden MVs. MVs and their TNF cargo were characterised by western blotting, confocal and immune-electron microscopy. These TNF+ve MVs were then instilled into the lungs of untreated mice, and after 4 hours their biological effects compared to BMDM-derived MVs harvested from TNF-/- mice (TNF-ve MVs). Recombinant TNF (100ng/ml, 50µL) was also instilled to compare the effects of MV-mediated vs soluble TNF signalling. Results Intriguingly, BMDMderived MVs carried the pro-TNF (26kDa) isoform rather than the soluble (17kDa) isoform, as assessed by western blotting. Immuneelectron/ confocal microscopy demonstrated that macrophages actively packaged pro-TNF within MVs during formation, which eventually localised to vesicle membranes. TNF+ve MVs induced significant intra-alveolar inflammation compared to TNF-ve MVs: lung inflammatory monocyte infiltration (410,700 ± 38,700 vs 214,000 ± 28,800 cells/ml, p<0.01, n=4); BALF protein (0.34 ± 0.03 vs 0.24 ± 0.01 mg/ml, p<0.05, n=4); ICAM-1 expression on epithelial cells (mean fluorescence intensity: 317 ± 36 vs 214 ± 14, p<0.05 n=4); and BALF KC levels (2746 ± 285 vs 1854 ± 134 pg/ml, p<0.05 n=4). Unlike the potent effects of MV-laden TNF, intratracheal soluble TNF did not produce significant inflammation, despite its dose exceeding the amount of proTNF in the MVs. Conclusion Our data demonstrate, for the first time, that danger signals (e.g. ATP) secrete TNF as the membrane pro-TNF isoform within MVs, inducing intra-alveolar inflammation (consistent with ALI) in a TNF-dependent manner. The soluble form of TNF (normally released within alveoli) could be quickly degraded/neutralised and would not reach target cells very effectively. In contrast, pro-TNF enclosed within MVs seems to be protected, allowing efficient longer-range signalling. Furthermore, we provide an alternative hypothesis for membrane TNF signalling in inflammatory lung disease (previously thought to occur only upon direct cell-to-cell contact): membrane TNF can be secreted within shed MVs, activating more-distant target cells without cell contact per se. : 
 
Description LUNG VOLUME REDUCTION FOR COPD MICROVESICLES study: 'Studying epithelial microvesicle production in patients treated with lung volume reduction for emphysema' 
Organisation Royal Brompton Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution The Royal Brompton Team perform bronchoscopies on patients with severe COPD, taking bronchoalveolar lavage fluid, blood and bronchial brushings. We then perform ELISAs on these samples as well as measure the Microvesicle numbers. The aim is to find a novel biomarker in these patients.
Collaborator Contribution The Royal Brompton Team sample the patients and perform detailed clinical examination/investigations in order to stage their disease severity.
Impact none as of yet
Start Year 2016
 
Description The Role of acid Sphingomyelinase in Acute lung Injury 
Organisation Charité - University of Medicine Berlin
Country Germany 
Sector Academic/University 
PI Contribution Microvesicle-mediated signalling during acute lung injury: Defining the role of acid sphingomyelinase Aim To investigate the role of acid sphingomyelinase (ASM) in microvesicle (MV) production during acute lung injury (ALI), and its clinical presentation acute respiratory distress syndrome (ARDS). Background Microvesicles in ARDS: Microvesicles (MVs), membrane-circumscribed extracellular particles of 100-1000nm in size, are mediators of cellular communication carrying a variety of molecular cargo over short and long distances to target cells1-3. We have identified that alveolar macrophage MVs are rapidly released during ALI (Figure 1) and that these MVs have important pathogenic roles in ALI, mediated by the molecular cargo enclosed within them4 5 (Figure 2). MV shedding occurs during inflammation in response to danger signals (e.g. extracellular ATP) or cellular stress (e.g. hypoxia). A common pathway to these insults is the translocation of ASM from lysosomes to the cell membrane, where it catalyses ceramide formation from sphingomyelin1 6, altering membrane physical properties, leading to MV formation and release (Figure 3)6. As key mediators of inflammation, MVs play a crucial role in the rapid secretion of cytokines which lack a secretory sequence (i.e. non-classically released proteins e.g. IL-1ß), and this has been shown to be dependent on ASM since IL-1ß secretion is substantially reduced from ASM -/- cells7-9. Interestingly, classically secreted cytokines e.g. TNF9, which have well-defined endoplasmic reticulum (ER)/Golgi-dependent trafficking pathways, have also been found within MVs2 4. Indeed we have demonstrated that danger signals (e.g. ATP) redirects intra-cellular TNF trafficking, from classical ER/Golgi-dependent pathway to a non-classical, ER/Golgi-independent route, secreting TNF within MVs in a similar fashion to IL-1ß5 (Figure 4), and that this switch in TNF secretion is also mediated by ASM (Figure 5). Acid sphingomyelinase and ARDS: ASM has been implicated in the pathophysiology of ALI/ARDS since ASM levels are increased within the lungs in models of ALI6 and ASM-/- mice appear to be protected from ALI10 11. It remains unclear why these animals are protected but is thought to be due to inhibition of ceramide production12, since ceramide initiates intra-cellular pro-inflammatory pathways and is elevated in bronchoalveolar lavage fluid (BALF) in patients with ARDS13. Although ceramide has been touted as a potential therapeutic target in ARDS12, its mechanistic role in ARDS must be carefully addressed prior to clinical translation. Importance and hypothesis Patients with ARDS have a high mortality and consume considerable healthcare resources due to severity of illness and long intensive care stays14. Despite intense research (28,000+ articles on PubMed), there are no disease-modifying therapies for ARDS. There remains an urgent, unmet need for a re-direction in ALI research to identify novel therapeutic targets, which may lead to effective treatments. Since MVs carry a milieu of inflammatory cargo (e.g. proteins, lipids, miRNAs), inhibiting MV-production would interrupt signalling of multiple inflammatory mediators contained within vesicles (rather than focussing on individual ones), and consequently MVs are an attractive target. Since our previous in vitro work suggests that ASM may play a role in MV generation and packaging of their cargo mediators (Figure 5), targeting ASM could lead to novel therapies inhibiting MV-mediated signalling in ALI. We hypothesise that ASM is activated in pulmonary cells following injurious stimuli and plays a crucial role in the pathophysiology of ALI/ARDS, by generating 'pro-inflammatory' MVs. These MVs may initiate and/or propagate ALI, thereby explaining why ASM-/- mice are protected in models of ALI10. Furthermore, we propose that ASM modulates protein trafficking through the generation of ceramide, which induces activation of intracellular pathways, regulating molecular cargo packaging within MVs and hence MV biological effects. We aim to investigate this hypothesis via 3 objectives: 1) To confirm in-vivo that ASM knockout (-/-) alveolar cells produce reduced number of MVs 2) To investigate the molecular cargo packaged within MVs produced from ASM -/- alveolar cells and identify whether ceramide is mechanistically involved in packaging cargo within MVs. 3) To determine the functional activity of MVs originated from ASM-/- alveolar cells and determine the significance of ASM in MV mediated signalling during ALI Research plan Aim 1: MV production in vivo We will use well-established clinically-relevant models of ALI induced by intratracheal lipopolysaccharide (LPS) (pathogen-related inflammation)15 and hydrochloric acid16 (sterile inflammation), to elucidate the patterns of MV release during the course of ALI from injury to resolution in wild-type and ASM -/- mice. MVs will be isolated from BALF as previously described4 and their numbers, cell source (e.g. neutrophil, macrophage) and phenotypes will be determined by flow cytometry using protocols developed during my PhD5 17. MV profiles will be correlated with physiological and immunological parameters of ALI, e.g. respiratory mechanics, oxygenation, pulmonary oedema, BALF/plasma cytokine levels and histology15 16. Having identified the kinetics of MV release in vivo, we will then develop in vitro models for mechanistic studies. We will harvest primary macrophages5 and neutrophils18 from either wild-type or ASM-/- mice and induce MV release in vitro using four different stimuli: 1) LPS; 2) LPS + ATP; 3) LPS + exogenous ASM; 4) LPS + exogenous ceramide. Sphingolipid/ceramide concentrations will be assessed via liquid chromatography and mass spectrometry (pre + post stimulation)19. Aim 2: Molecular cargo within MVs MVs will be collected from in-vitro and in-vivo models using both wild-type ASM -/- cells as described above, and analysed for their intra-vesicular mediators by ELISA/western blotting/confocal microscopy, focussing upon classically released cytokines e.g. TNF and non-classically released cytokine e.g. IL-1ß. Since ASM may exert its effects through ceramide, MVs produced from wild-type cells in the presence of ceramide-specific antibodies12 will be analysed for their molecular cargo. We will also use live cell confocal imaging to assess the effect of ceramide on TNF and IL-1 release. Aim 3: Biological activity of MVs To assess functional activity of MVs produced from cells devoid of ASM, MVs from wild-type and ASM -/- cells will be intratracheally instilled into mice and activation status of intra-alveolar cells/parameters of ALI will be determined. Finally, we will try to inhibit MV-mediated signalling by targeting ASM in vivo. We will administer pharmacological inhibitors of ASM (e.g. Desipramine, Figure 5) to mouse models of ALI described in Aim 1 and assess whether pharmacological inhibition of ASM can protect against ALI. References 1. Soni S, Tirlapur N, O'Dea KP, Takata M, Wilson MR. Microvesicles as new therapeutic targets for the treatment of the acute respiratory distress syndrome (ARDS). Expert opinion on therapeutic targets 2019;23(11):931-41. 2. Kunder CA, John ALS, Li G, Leong KW, Berwin B, Staats HF, Abraham SN. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. Journal of Experimental Medicine 2009;206(11):2455-67. 3. O'Dea KP, Tan YY, Shah S, V Patel B, C Tatham K, Wilson MR, Soni S, Takata M. Monocytes mediate homing of circulating microvesicles to the pulmonary vasculature during low-grade systemic inflammation. Journal of Extracellular Vesicles 2020;9(1):1706708. 4. Soni S, Wilson MR, O'dea KP, Yoshida M, Katbeh U, Woods SJ, Takata M. Alveolar macrophage-derived microvesicles mediate acute lung injury. Thorax 2016:thoraxjnl-2015-208032. 5. Soni S, O'Dea KP, Tan YY, Cho K, Abe E, Romano R, Cui J, Ma D, Sarathchandra P, Wilson MR. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles. The FASEB Journal 2019:fj. 201802386R. 6. Verderio C, Gabrielli M, Giussani P. Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles. Journal of lipid research 2018;59(8):1325-40. 7. Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E. Acid sphingomyelinase activity triggers microparticle release from glial cells. The EMBO journal 2009;28(8):1043-54. 8. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, Surprenant A. Rapid secretion of interleukin-1ß by microvesicle shedding. Immunity 2001;15(5):825-35. 9. Murray RZ, Stow JL. Cytokine secretion in macrophages: SNAREs, Rabs, and membrane trafficking. Frontiers in immunology 2014;5 10. Peng H, Li C, Kadow S, Henry BD, Steinmann J, Becker KA, Riehle A, Beckmann N, Wilker B, Li P-L. Acid sphingomyelinase inhibition protects mice from lung edema and lethal Staphylococcus aureus sepsis. Journal of molecular medicine 2015;93(6):675-89. 11. von Bismarck P, García Wistädt C-F, Klemm K, Winoto-Morbach S, Uhlig U, Schutze S, Adam D, Lachmann B, Uhlig S, Krause MF. Improved pulmonary function by acid sphingomyelinase inhibition in a newborn piglet lavage model. American journal of respiratory and critical care medicine 2008;177(11):1233-41. 12. Göggel R, Winoto-Morbach S, Vielhaber G, Imai Y, Lindner K, Brade L, Brade H, Ehlers S, Slutsky AS, Schütze S. PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide. Nature medicine 2004;10(2):155. 13. Rauvala H, Hallman M. Glycolipid accumulation in bronchoalveolar space in adult respiratory distress syndrome. Journal of lipid research 1984;25(11):1257-62. 14. Matthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, Herridge M, Randolph AG, Calfee CS. Acute respiratory distress syndrome. Nature Reviews Disease Primers 2019;5(1):18. 15. Oakley C, Koh M, Baldi R, Soni S, O'Dea K, Takata M, Wilson M. Ventilation following established ARDS: a preclinical model framework to improve predictive power. Thorax 2019;74(12):1120-29. 16. Patel BV, Wilson MR, Takata M. Resolution of acute lung injury and inflammation: a translational mouse model. European Respiratory Journal 2012;39(5):1162-70. 17. Soni S. Microvesicles are key mediators of inflammation in acute lung injury. Imperial College London, 2018. 18. Karmakar M, Katsnelson MA, Dubyak GR, Pearlman E. Neutrophil P2X 7 receptors mediate NLRP3 inflammasome-dependent IL-1ß secretion in response to ATP. Nature communications 2016;7(1):1-13. 19. McVey MJ, Kim M, Tabuchi A, Srbely V, Japtok L, Arenz C, Rotstein O, Kleuser B, Semple JW, Kuebler WM. Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets. American Journal of Physiology-Lung Cellular and Molecular Physiology 2017;312(5):L625-L37.
Collaborator Contribution as above
Impact European Respiratory Society Research Fellowship Award
Start Year 2020
 
Description American Thoracic Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract:
Rationale TNF plays a key role in inflammatory lung diseases including acute lung injury (ALI). We previously demonstrated the presence of TNF
within microvesicles (MVs) released in the alveoli during a LPS-induced model of ALI. Although MVs form an ideal vehicle for ferrying 'non-classically
secreted' cytokines (e.g. Il-1ß/IL-18), it is unknown why TNF, a 'classically secreted' cytokine, is released within MVs. Here we investigate the
hypothesis that MV-mediated TNF secretion allows protected, more efficient TNF signalling compared to its soluble counterpart. Methods Bone
marrow derived macrophages (BMDMs) were harvested from C57/BL6 mice, stimulated with LPS and then ATP to generate TNF-laden MVs. MVs and
their TNF cargo were characterised by western blotting, confocal and immune-electron microscopy. These TNF+ve MVs were then instilled into the
lungs of untreated mice, and after 4 hours their biological effects compared to BMDM-derived MVs harvested from TNF-/- mice (TNF-ve MVs).
Recombinant TNF (100ng/ml, 50µL) was also instilled to compare the effects of MV-mediated vs soluble TNF signalling. Results Intriguingly, BMDMderived
MVs carried the pro-TNF (26kDa) isoform rather than the soluble (17kDa) isoform, as assessed by western blotting. Immuneelectron/
confocal microscopy demonstrated that macrophages actively packaged pro-TNF within MVs during formation, which eventually localised
to vesicle membranes. TNF+ve MVs induced significant intra-alveolar inflammation compared to TNF-ve MVs: lung inflammatory monocyte
infiltration (410,700 ± 38,700 vs 214,000 ± 28,800 cells/ml, p<0.01, n=4); BALF protein (0.34 ± 0.03 vs 0.24 ± 0.01 mg/ml, p<0.05, n=4); ICAM-1
expression on epithelial cells (mean fluorescence intensity: 317 ± 36 vs 214 ± 14, p<0.05 n=4); and BALF KC levels (2746 ± 285 vs 1854 ± 134 pg/ml,
p<0.05 n=4). Unlike the potent effects of MV-laden TNF, intratracheal soluble TNF did not produce significant inflammation, despite its dose
exceeding the amount of proTNF in the MVs. Conclusion Our data demonstrate, for the first time, that danger signals (e.g. ATP) secrete TNF as the
membrane pro-TNF isoform within MVs, inducing intra-alveolar inflammation (consistent with ALI) in a TNF-dependent manner. The soluble form of
TNF (normally released within alveoli) could be quickly degraded/neutralised and would not reach target cells very effectively. In contrast, pro-TNF
enclosed within MVs seems to be protected, allowing efficient longer-range signalling. Furthermore, we provide an alternative hypothesis for
membrane TNF signalling in inflammatory lung disease (previously thought to occur only upon direct cell-to-cell contact): membrane TNF can be
secreted within shed MVs, activating more-distant target cells without cell contact per se.
Year(s) Of Engagement Activity 2018
 
Description BSc presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Informing students of microvesicles, their role in acute lung injury and our groups results
Year(s) Of Engagement Activity 2016
 
Description BSc presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact To publicise the lab's research
Year(s) Of Engagement Activity 2016,2017
 
Description British Association of Lung Research Annual Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Introduction
Microvesicles (MVs) are important mediators of inter-cellular communication within the alveolar space, causing significant alveolar epithelial cell (AEC) injury, and key components in the pathophysiology of lung inflammation such as acute respiratory distress syndrome (ARDS). Yet, it remains unclear how MVs interact/signal with target cells in the alveolus.

Methods
Macrophage- or murine lung epithelial cell (MLE)-derived MVs were labelled with membrane intercalating dye DiD, and incubated for 1-4h with coculture of primary alveolar macrophages (AM) and MLE cells, with/without LPS priming. In vivo-generated MVs (a mixed population consisting mainly of AM/AEC-derived MVs harvested from intratracheal (i.t.) LPS treated mice) were DiD-labelled and i.t. instilled into mice for 1h, with/without LPS pre-treatment. Cellular MV uptake was evaluated by flow cytometry, using cell surface markers and DiD mean fluorescence intensity (MFI).

Results
In vitro, the majority of MVs were taken up by AMs compared to MLE cells, irrespective of MV cell sources or LPS priming. In vivo, preferential uptake by AMs vs AEC was also observed: for macrophage-derived MVs, AM MFI 1153±297.6 vs AEC MFI 4.53±2, p<0.05 n= 3; for in vivo-derived MVs, AM MFI 1397±139 vs AEC MFI 11.59±2, p<0.001 n=5. In LPS-pretreated mice, this pattern did not change.

Conclusions
We found that MV uptake/internalisation was performed predominantly by AMs, irrespective of MV phenotype under resting/inflamed conditions. Our results strongly suggest that alveolar macrophages play a crucial, previously unappreciated role in modulating MV processing within the alveoli, potentially regulating MV-mediated intra-alveolar inflammation in the pathophysiology of diseases such as ARDS.
Year(s) Of Engagement Activity 2019
 
Description European Respiratory Society Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact To inform peer involved in my field of research of our findings
Year(s) Of Engagement Activity 2015
 
Description European Respiratory Society Oral Presentation of my work 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Background: Microvesicles (MV) are important mediators of inter-cellular communication within the alveolar
space, causing significant alveolar epithelial cell (AEC) injury (Soni et al. Thorax 2016;71:1020-29). Here
we investigate, for the first time, MV uptake profile by individual intra-alveolar cells during resting/inflamed
lung conditions.
Methods: Macrophage- or murine lung epithelial cell (MLE)-derived MVs were labelled with membrane
intercalating dye DiD, and incubated for 1-4h with coculture of primary alveolar macrophages (AM) and
MLE cells, with/without LPS priming. AM-derived or in vivo-generated MVs (a mixed population consisting
mainly of AM/AEC-derived MVs harvested from intratracheal (i.t.) LPS treated mice) were DiD-labelled and
i.t. instilled into mice for 1h, with/without LPS pre-treatment. Cellular MV uptake was evaluated by flow
cytometry, using cell surface markers and DiD mean fluorescence intensity (MFI).
Results: In vitro, the majority of MVs were taken up by AMs compared to MLE cells, irrespective of MV cell
sources or LPS priming. In vivo, preferential uptake by AMs vs AEC was also observed: for AM-derived
MVs, AM MFI 1175±544 vs AEC MFI 8±2, p<0.05 n= 3; for in vivo-derived MVs, AM MFI 1598±166 vs AEC
MFI 15±2, p<0.001 n=3. In LPS-pretreated mice, this pattern did not change.
Conclusion: Surprisingly, and in contrast to a previous report of significant MV uptake by AEC (Bourdonnay
et al. J Exp Med 2015;212:729-42), we found that MV uptake/internalisation was performed almost
exclusively by AMs, irrespective of MV phenotype under both resting and inflamed conditions. The results
strongly suggest that AMs play a crucial modulating role in MV-mediated intra-alveolar inflammation.
Year(s) Of Engagement Activity 2017
 
Description How to get into Anaesthetic Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Details about my research and how to get involved with research in the future
Year(s) Of Engagement Activity 2017
 
Description ICS State of the Art: he Future Generation of Researchers - The Gold Medal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Acute lung injury (ALI) and its clinical presentation Acute Respiratory Distress Syndrome (ARDS) have an unacceptably high mortality1,2. Treatment remains principally supportive3 despite a vast amount of clinical/pre-clinical research. Therefore, a re-direction in ALI research is required, moving away from highlighting individual inflammatory mediators and instead investigating how inflammatory cargo are transmitted between cells. Microvesicles (MVs) are membrane-circumscribed extracellular particles (size: 100-1000nm), providing an alternative yet crucial role in intercellular communication by carrying a variety of bioactive cellular cargo4-8. However, their role in ALI/ARDS remains unknown. We hypothesised that MVs are key to the pathogenesis of ALI as mediators of intercellular communication and our aims were to examine: the role of MVs in the pathophysiology of ALI; the inflammatory mediators carried by alveolar MVs; how MVs communicate with target alveolar cells; and characterise MVs in patients with severe inflammatory lung disease.

We used various in vitro/in vivo techniques and developed a number of intricate experimental protocols (using flow cytometry, ELISA, western blotting, confocal microscopy, immune-electron microscopy) to fully characterise/phenotype MVs. We identified MVs populations in bronchoalveolar lavage fluid (BALF) taken from a lipopolysaccharide-induced murine model of ALI. Primary alveolar macrophages/bone marrow-derived macrophages were harvested from untreated mice and stimulated with lipopolysaccharide (1µg/ml) and ATP (3mM) to produce MVs. To assess their inflammatory activity, these MVs were incubated with epithelial cells in vitro or instilled intratracheally into mice. To ascertain MV-mediated communication within the alveolus, labelled MVs were instilled intratracheally into the lungs of mice or incubated within a co-culture of primary alveolar macrophages and epithelial cells (under inflammatory/anti-inflammatory conditions). In addition, pharmacological/molecular inhibitors were used to identify mechanisms of MV-mediated communication with alveolar cells. Finally, 61 patients with severe chronic obstructive pulmonary disease (COPD) underwent rigorous clinical evaluation using symptom/lung function/severity scores. BALF/blood samples were obtained, analysed for MVs and MV counts were correlated to clinical markers/indices of disease severity.

Initially we profiled MV populations produced during ALI. We demonstrated for the first time, that MVs from different intra-alveolar precursor cells are sequentially produced within the alveolar space early in the course of ALI. We focussed upon alveolar macrophage-derived MVs since they were the dominant population and found they packaged significant amounts of TNF rather than other pro-inflammatory material (e.g. IL-1ß/IL-6). These alveolar macrophage-derived MVs increased epithelial cell ICAM-1 expression, via a TNF dependent mechanism (p<0.001). When instilled intratracheally into mice, these MVs induced changes consistent with ALI e.g. increased BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05). Therefore these alveolar macrophage-derived MVs, are potent initiators of ALI, mediated by their molecular cargo, particularly TNF and these results highlight that MVs are key components in the pathophysiology of ALI/ARDS.

Following this, we examined how cells packaged TNF into MVs. In severe sepsis/ARDS, substantive cell injuries occur with subsequent release of danger signals (e.g. ATP)9,10, inducing MV production11,12. We discovered that ATP totally redirects TNF trafficking, inhibiting soluble TNF release from cells, instead preferentially packaging the membrane TNF isoform into potent, pro-inflammatory MVs. Within MVs, TNF is protected from extracellular degradation, dispersion or neutralisation. Using pharmacological/genetic inhibitors, we discovered that TNF secretion within MVs bypasses the conventional endoplasmic reticulum/Golgi-dependent pathway (despite being a classically-released cytokine), and is mediated by acid sphingomyelinase. Unlike TNF+ve MVs, TNF-ve MVs (harvested from TNF knockout mice) did not produce inflammation in vivo (assessed using BALF inflammatory cells, protein, CXCL1 and epithelial cell ICAM-1 expression (p<0.05)) highlighting the physiological importance TNF enclosed within MVs. These data, offer important, previously unappreciated clinical implications. Clinical trials of anti-TNF therapy have shown little beneficial impact in sepsis/ARDS, despite ample preclinical evidence of TNF involvement in their pathophysiology13. In severe sepsis/ARDS, substantial ATP release takes place, which would lead to secretion of TNF in MVs (rather than soluble form). Antibody-based blocking treatments may not be effective to inhibit the effects of TNF enclosed within MVs. Our study suggests that targeting MV-mediated TNF signalling (by reducing MV uptake/production), may be essential to block TNF biological effects in sepsis/ARDS.









We next investigated how MVs communicate with target cells within the alveolar space, that although both epithelial cells and alveolar macrophages take up MVs, alveolar macrophages take up the overwhelming majority, regardless of the environmental condition (p<0.001). We demonstrated that epithelial cell uptake is dependent on integrin/phosphatidylserine binding whilst alveolar macrophage uptake is more dependent on scavenger receptors indicating clear mechanistic differences between these cell types. Our results strongly suggest that alveolar macrophages play a dominant, crucial role in modulating MV processing within the alveoli, regulating MV-mediated intra-alveolar inflammation in the pathophysiology of ALI. These data highlight future areas of study by which researchers can modulate the actions of MVs within the alveolar space.









Finally, we consolidated and translated our in vitro/in vivo data by characterising different MV populations in 61 patients with severe COPD. We confirmed the presence of multiple MV populations within BALF including leukocyte, neutrophil, monocyte, alveolar macrophage and epithelial MVs. We assessed whether these BALF MV populations could be potential biomarkers for COPD and found that BALF neutrophil MVs strongly correlated with a broad range of indices of disease severity including symptoms (MRC dyspnoea score) (p<0.01); exercise tolerance (6 minute walk test) (p<0.01); disease severity (FEV1/RV/TLC) (p<0.05); exacerbation frequency (p<0.01) and mortality (BODE index) (p<0.01). We demonstrated BALF neutrophil-derived MV numbers correlate with functional and clinically relevant disease severity indices. These data strongly corroborate our in vitro/in vivo data and suggest that BALF neutrophil MVs are a novel, clinically relevant biomarker for COPD disease severity, and may have a pathogenic role in COPD progression.

In conclusion, we have demonstrated a number of breakthrough findings which have considerable translatable and clinical significance. Our findings provide substantial evidence that MVs are key components in the pathophysiology of lung inflammation/ARDS. Furthermore in these clinical scenarios, where there is a distinct lack of biomarkers, MVs are indicators of disease severity that could be used in clinical practice. Finally, MVs are disease-modifiable targets and our studies have pioneered novel diagnostic and therapeutic perspectives in patients with ARDS, where no curative treatment currently exists.

We would like to thank the Medical Research Council and the British Journal of Anaesthesia for funding this work.
Year(s) Of Engagement Activity 2018
 
Description MRes Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact To present the research of the department
Year(s) Of Engagement Activity 2016,2017
 
Description MRes presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Discussed the project outputs with postgraduate students and tried to generate discussion and increase interest
Year(s) Of Engagement Activity 2015
 
Description MRes teaching 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact How to prepare an abstract for conference
Year(s) Of Engagement Activity 2017
 
Description Perioperative Trainee Research Forum Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact To increase the visibility of the research in our lab/across london in the field of anaesthesia
Year(s) Of Engagement Activity 2016
 
Description Talk at ICU at Chelsea and Westminster Hospital 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact To inform clinicians of the basic science work carried out at Imperial College
Year(s) Of Engagement Activity 2015
 
Description Talk at NIHR CRN Wessex and Thames Valley Urgent Care Research event. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Background: TNF plays a key role in inflammatory lung diseases including acute
lung injury (ALI). We previously demonstrated the presence of TNF within
microvesicles (MVs) released during a LPS-induced model of ALI. It is unknown why
cells release TNF within MVs.
Aims: Investigate our hypothesis that MV-mediated TNF secretion allows protected,
more efficient TNF signalling compared to its soluble counterpart.
Methods: Macrophages were harvested from mice, stimulated to generate TNFladen
MVs (LPS and ATP) and characterised by western blotting/confocal/immuneelectron
microscopy. These TNF+ve MVs were instilled into the lungs of untreated
mice, and their biological effects compared to TNF-ve MVs (harvested from TNF-/-
mice). Recombinant TNF was also instilled to compare the effects of MV-mediated vs
soluble TNF signalling.
Results: Macrophage-derived MVs carried the pro-TNF (26kDa) isoform rather than
the soluble (17kDa) isoform (assessed by western blotting). Immuneelectron/
confocal microscopy demonstrated that macrophages actively packaged
pro-TNF within MVs during formation. TNF+ve MVs induced significant intra-alveolar
inflammation compared to TNF-ve MVs (n=4): lung inflammatory monocyte infiltration
(p<0.01); BALF protein (p<0.05); ICAM-1 expression on epithelial cells (p<0.05); and
BALF cytokine levels (p<0.05). Unlike the potent effects of MV-laden TNF, soluble
TNF did not produce significant inflammation.
Conclusions: Our data demonstrate, for the first time, that cells package pro-TNF
within MVs, inducing intra-alveolar inflammation (consistent with ALI) in a TNFdependent
manner. Pro-TNF enclosed within MVs seems to be protected compared
to soluble TNF, allowing efficient longer-range signaling. These data have crucial
biological implications for this key pro-inflammatory cytokine, particularly when
therapeutically targeting TNF in ARDS.
Year(s) Of Engagement Activity 2018
 
Description Undergraduate BSc teaching 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Explanation about role of microvesicles in ARDS
Year(s) Of Engagement Activity 2017