Extracellular vesicles : mediators of immune modulation in the lung and as therapeutic vehicles

Extracellular vesicles (EVs) are released from all cell types, and carry a wide setup of proteins, nucleic acids, lipids and other cargo. The overall aim of this thesis is to explore EV-based immune therapy, but also to find clues on mechanisms of the inflammatory disease sarcoidosis, and of lung cancer. Exosomes from dendritic cells (DCs) pulsed with antigen can induce antigen-specific responses in vitro and in vivo. Study I is an investigation comparing exosomes and microvesicles (MVs), which may complement exosomes therapeutically. We found surprisingly similar phenotypes of the two EV subtypes, including size distribution and immunestimulatory molecule expression. However, when tested in vivo, only exosomes induced a significant antigen-specific CD8+ T cell response. Antigenic restimulations ex vivo did, however, suggested that also MVs had such capacity, and both vesicle types induced antigen-specific IgG production. We further targeted the inflammatory disease pulmonary sarcoidosis in study II and study III with the aim to increase understanding of disease mechanisms, but also to search for disease biomarker candidates, and possible new treatment regimens. Broad proteomic characterizations of exosomes from patients revealed high abundance of proinflammatory molecules including leukotriene (LT)-forming enzymes. Large portions of the complement system were elevated, and we flagged vitamin Dbinding protein as a possible biomarker for sarcoidosis. Functional tests of patient exosomes further suggested that they can engage monocytes and favor release of pro-inflammatory cytokines. The effects were partly dependent on LTs, and we could reduce cytokine production using the commercially available LT receptor antagonist Montelukast. Study IV on exosomes in lung cancer is focused on how exosomes may contribute to tumor progression via LTs. Exosomes from pleural effusions of patients favored generation of tumorigenic LTD4, as well as tumor cell migration, which could be reduced using Montelukast. In summary, this thesis highlights the importance of investigating all EV subtypes in both basic and applied research. Further, the ability of EVs to contribute to inflammatory processes in the lungs underscores the potential of EVs in understanding disease mechanisms and finding diagnostic and prognostic disease markers. Finally, all three lung studies IIIV point to the possibility of interfering with LTs in inflammatory conditions, with possible applications also in cancer therapy

Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) – a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis

Plasma levels of platelet-derived microvesicles are associated with risk of future venous thromboembolism

Background - Microvesicles (MVs) are small double‐membrane encapsulated particles shed from cells. Case‐control studies have reported elevated plasma levels of platelet‐derived MVs (PDMVs) in patients with venous thromboembolism (VTE). However, it is not known whether high PDMV levels is a risk factor or a consequence of the acute VTE event. Objectives - To investigate the association between PDMVs in plasma and risk of future incident VTE. Methods - We performed a population‐based nested case‐control study with 314 VTE cases and 705 age‐ and sex‐matched controls (from The Tromsø Study) to investigate the association between the proportion of PDMVs (PDMVs%) in plasma and risk of future incident VTE. MVs isolated from plasma sampled at baseline (i.e., before VTE) were stained for platelet markers and analyzed by flow cytometry. PDMVs% were defined as the number of PDMVs divided by the total number of MVs. Odds ratios (ORs) with 95% confidence intervals (CI) for VTE risk were estimated across quartiles of PDMVs%. Results - Subjects with PDMVs% in the highest quartile had an OR for VTE of 1.78 (95% CI: 1.21–2.64) and 1.99 (95% CI: 1.24–3.26) for provoked VTE, compared to those in the lowest quartile. The association was moderately affected by multivariable adjustment for age, sex, body mass index, C‐reactive protein, platelet count, and cancer. The OR for VTE was higher when the time between blood sampling and event was shorter. Conclusions - Our results show that high proportions of PDMVs are associated with future risk of incident VTE and imply a role of platelet activation in the pathogenesis of VTE