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Title: Microvesicles are key mediators of inflammation in acute lung injury
Author: Soni, Sanooj
ISNI:       0000 0004 7969 856X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Acute lung injury (ALI) and its clinical presentation Acute Respiratory Distress Syndrome (ARDS) has an unacceptably high mortality, surpassing 40% in those with the severest form of the disease. Despite intensive research activity and the identification of a vast number of biological mediators, all of which have been promoted as apparently vital to the pathogenesis of ALI/ARDS, treatment remains principally supportive. 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 of 100-1000nm in size and are derived from eukaryotic cells following direct damage, apoptosis or stimulation. MVs provide an alternative yet crucial role in intercellular communication by carrying a variety of bioactive cellular cargo and have been implicated in the pathophysiology of various inflammatory diseases. Yet, their role in acute lung injury (ALI) remains unknown. We hypothesise that MVs are key to the pathogenesis of ALI as mediators of intercellular communication and our aim in this thesis was to assess the role of MVs in ALI. We demonstrated that there is a dynamic production of MVs from multiple intra-alveolar cells very early during the inflammatory course of ALI with a substantial increase in MVs originating from alveolar macrophages. When instilled into the lungs of mice, these alveolar macrophage-derived MVs significantly increased the parameters of ALI tested, providing compelling evidence for their role in the pathophysiology of ARDS. We found that these MVs packaged TNF initiating inflammation via a TNF dependent mechanism. Mechanistically, we found that TNF is packaged within MVs, in a similar fashion to IL-1β, via an unconventional, ER/Golgi-independent route, despite being a classical cytokine (Golgi-dependant). Using pharmacological and genetic inhibition techniques, we demonstrated that this switch in cellular TNF trafficking is mediated via acid sphingomyelinase. . Furthermore, we established that the TNF within MVs, is the transmembrane TNF isoform (rather than soluble isoform) and localises to the MV membrane in a stable manner, activating distant target cells without cell-to-cell contact per se. Therefore we identified an entirely novel way in which membrane TNF signals in vivo. Next we examined how MVs communicate within the alveolar space. We showed that although both epithelial cells and alveolar macrophages take up MVs, alveolar macrophages take up the overwhelming majority, regardless of the environmental condition. We demonstrate that epithelial cell uptake is dependent on integrin/phosphatidylserine binding while alveolar macrophage uptake is more dependent on scavenger receptors indicating clear mechanistic differences between these cell types. Our results suggested two potential scenarios regarding MV-mediated communication within the alveolus: 1) Activation of epithelial cells may be due to TNF enclosed within MVs, which binds to TNF receptors on the surface of epithelial cells (rather than internalisation_; or 2) ICAM-1 upregulation may be assisted by alveolar macrophages, potentially by 'passing' on proinflammatory material to epithelial cells. We consolidated and translated the findings from our in vitro and in vivo models of ALI/ARDS by characterising different MV populations within 30 patients with another inflammatory lung disease, Chronic Obstructive Pulmonary Disease (COPD). We confirmed the presence of multiple MV populations within bronchoalveolar lavage fluid (BALF) taken from our patients (e.g. leukocyte, neutrophil, monocyte, alveolar macrophage, epithelial). We assessed whether these BALF MV populations could be potential biomarkers for COPD and found that BALF neutrophils strongly correlated with a broad range of indices of disease severity including symptoms (MRC dyspnoea score, St George's Respiratory Questionnaire); exercise tolerance (6 minute walk test); disease severity (FEV1); and mortality (BODE index). Taken together, our findings support a role for MVs in the pathogenesis of ALI and lung inflammation. Furthermore MVs are biomarker of lung inflammation that could be used in clinical medicine. Therefore modulation of MVs within the alveolus may be a promising therapeutic target and our studies may open novel diagnostic and therapeutic perspectives in patients with ARDS.
Supervisor: Takata, Masao Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral