Abstract
Extracellular vesicles (EV) are membrane encapsulated nanoparticles that are released by all cell types. EV have been detected in all body fluids, including blood and semen. EV transfer their constituents, including cytosolic- and membrane proteins, lipids and RNA molecules, from their producing cells to acceptor cells. In this capacity, EV
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function as intercellular signaling devices in many physiological and pathophysiological processes, such as immune regulation, cancer, and sperm cell activation. Moreover, EV in body fluids hold great potential to be used as biomarkers for disease. In addition to EV, body fluids contain multiple other particles with biological activities, including lipoprotein particles and protein complexes. This imposes great challenges for isolating EV from blood. In the first part of my thesis, we developed a novel unbiased procedure, consisting of three sequential steps: precipitation by polyethylene glycol, floatation by ultracentrifugation into iohexol density gradients, and size exclusion chromatography, to isolate EV from blood or cell culture medium with both high yield and purity. The second part of my thesis involves EV from seminal plasma (spEV). spEV are thought to have immune regulatory functions to tolerize maternal immune cells within the female reproductive tract for semen, and in case of successful fertilization also for paternal antigens that are expressed by the fetus. We isolated two populations of spEV that differ in size and protein composition. Gene ontology enrichment analysis suggests that these two spEV subtypes are generated by distinct cellular pathways and molecular machineries. Interestingly, proteins that are exclusively or predominantly expressed by the prostate, and are either up or down-regulated in prostate cancer tissue are present in spEV. More studies are required to investigate the potential of these proteins to be used as EV associated prostate cancer biomarkers in blood. Interestingly, spEV also contain proteins with immune regulatory functions. Their presence indicates a role for spEV in immune regulation. We found that spEV interfered with in vitro differentiation of CD1a+ monocyte derived dendritic cells (moDC), suggesting interference with cell-mediated immunity. Moreover, spEV strongly inhibited the production of IFN-γ and TNF by isolated activated T cells, and reduced their surface expression of CD25. Additionally, spEV inhibited proliferation of activated T cells and strongly stimulated the expression of Foxp3 by activated CD4+ T cells, indicating Treg differentiation. We also found that spEV interfered with activation of TLR7 challenged plasmacytoid dendritic cells (pDC), as measured by decreased IFN-α secretion and CD40 expression. Interaction of CD38 on spEV with CD31 on pDC is involved in the tolerizing effect of spEV on pDC. These data indicate that spEV have a capacity to tolerize the adaptive immune system at multiple levels: spEV act directly on antigen presenting cells, including moDC and pDC, also on T cells. spEV may drive immune tolerance for allogeneic paternal antigens within the female reproductive tract. As a side effect, however, by inhibiting immune cell functions, spEV may promote virus transmission at coitus and prostate cancer. More experimentation is required to determine the precise molecular mechanism by which spEV interfere with immune cell functions.
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