Susanne Van Der Grein

Dutch Summary for Laypeople

Extracellular vesicles provide communication between cells
Communication between the cells in our body is essential for controlling various organ systems, including the immune system that protects us against pathogens. One of the methods by which cells communicate with each other is by exchanging packages containing signal molecules. These packages are surrounded by a membrane consisting of a double layer of lipids (fats), and are therefore also called extracellular (= outside the cell) membrane vesicles, or simply ‘vesicles’. These extracellular vesicles (EV) have a diameter of 0.05 – 1 micrometer (a cell = 10 – 100 micrometers) and are secreted by almost all cell types in the body. Additionally, EVs are present in various body fluids, including blood plasma, urine, and milk. EVs secreted by one cell can be taken up by another cell located nearby or at a distance. In this way, cells can send messages to each other. These messages are formed by different types of molecules loaded into the EVs, such as lipids, proteins, and RNA, a carrier of genetic material similar to DNA. In the cell that receives the EV, these molecular messages are ‘read’, and adjustments to various cellular processes can be made if necessary. The function of EVs as a cellular communication tool was discovered relatively recently. Since then, a role for EVs has been established in an increasing number of processes underlying health and disease. For example, EVs can perform various functions during infections with pathogens such as bacteria, parasites, and viruses. However, many questions remain about the role EVs play in inter-cellular communication and how this communication is regulated.

Various factors contribute to the heterogeneity of EV populations
The term ‘EV’ is used for a very diverse population of membrane vesicles that can vary greatly in physical properties, such as size and density. Additionally, the molecular composition of EVs can differ significantly. Because this molecular composition determines the effects EVs have on recipient cells, the diversity in EV content leads to a wide variation in possible functions. This diversity in phenotype (appearance), molecular content, and function is described as ‘heterogeneity’. Central to this thesis is the mapping of EV population heterogeneity, how molecular content relates to function, and which factors cause this heterogeneity.

A large number of factors can contribute to EV heterogeneity. For instance, different cell types secrete EVs with different contents and functions. EVs found in blood plasma may be secreted by red or white blood cells, or by cells lining the blood vessel walls. Beyond differences between cell types, significant variation can also occur within the population of EVs secreted by a single cell type. This is because cells can adapt to changing environmental conditions. Different oxygen levels, varying nutrient availability, and the presence of a pathogen can lead to a different ‘activation status’. An activated cell can secrete EVs with a different molecular composition and function than the same cell at rest. Finally, even a single cell type in a specific activation state can produce different subtypes of EVs. These subtypes can be formed via different routes. ‘Microvesicles’ are formed by budding and pinching off from the cell membrane. ‘Exosomes’ are formed in the endosome, a compartment within the cell. These vesicles are released into the extracellular space when the endosome fuses with the cell membrane. Current techniques cannot distinguish between these EV subtypes once they are outside the cell, which is why the collective term ‘EV’ is used to refer to all types of membrane vesicles.

Recently, another route for EV formation has been described involving ‘autophagy’. Autophagy is a process in which a cell encloses internal structures in a double-membrane compartment (the autophagosome) to break them down and recycle the building blocks. While it was long thought that autophagosomal contents were primarily delivered to the lysosome for degradation, it has recently been discovered that this material can also be secreted in EVs, a process called ‘secretory autophagy’.

In this thesis
Different sources of heterogeneity can cause large variations in EV phenotype, composition, and function. In this thesis, we investigated EV population heterogeneity at various levels in two different biological systems. First, we examined the heterogeneity caused by differences in the activation status of EV-producing cells, specifically looking at EVs secreted by dendritic cells (DC), which are crucial regulators of the immune system. Second, we studied heterogeneity within an EV population secreted by a single cell type under specific conditions, namely cells infected with naked viruses. In both systems, insight into the complexity of EV populations is vital for eventually answering questions about the function of EVs secreted by immune cells and their role during viral infections.

Heterogeneity in EV populations secreted by DC
DCs present antigens (molecules from pathogens) to T cells, which carry out immune responses to clear the pathogens. By responding to environmental signals, DCs can develop into cells that activate the immune system or cells that suppress it when no danger is present. These immune-suppressing DCs are also called tolerogenic DCs. Scientists have discovered that EVs secreted by immune-stimulating DCs can play a role in their immune-activating function. Much less is known about the function of EVs secreted by tolerogenic DCs. Knowledge of their potential immune-suppressive effects is particularly important for developing therapies for autoimmune diseases, where the immune system is overactive.

In Chapter 2, we investigated how different immune stimuli (activating vs. suppressive) influence the RNA content of EVs secreted by DCs. We cultured DCs with LPS (an activating component of bacteria) or with Vitamin D3 (a suppressive agent). We show that EVs from activating LPS-DCs contain different micro-RNAs than EVs from tolerogenic VitD3-DCs. In Chapter 3, we studied the functional consequences of these changes in RNA composition. We show that EVs from tolerogenic DCs can inhibit the release of the inflammatory cytokine IL-17 by a specific type of T cell called a ‘central memory’ T cell. We also discuss the potential link between the RNA content of these EVs and their function.

Heterogeneity in EV populations secreted by virus-infected cells
Viruses are small infectious particles that can only replicate within another organism (the host). Scientists have traditionally divided viruses into those that are or are not surrounded by a membrane. Viruses without a membrane are called ‘naked viruses’. They consist only of genetic material (RNA or DNA) protected by proteins. It was long thought that these viruses could only escape their host cell by causing it to burst (lysis). Recently, however, it was discovered that these viruses can also escape from intact cells by being enclosed in a membrane derived entirely from host material. These particles can be seen as EVs containing viruses. This discovery generated great interest because membrane enclosure significantly impacts how the virus and host interact. For example, the membrane can act as an ‘invisibility cloak’, preventing the virus from being recognized by the host's immune system.

In addition to the secretion of virus-containing EVs, viral infection can have other effects on EV release. Infection can lead to major changes in a cell, resulting in secreted EVs with different molecular compositions and functions. EVs from virus-infected cells have been described as having both pro-viral effects (promoting infection) and antiviral effects (counteracting infection). The effect is determined by the type and molecular composition of the EV. However, little is known about the heterogeneity of EV composition in naked virus infections. Chapter 4 provides an extensive review of various aspects of EV heterogeneity in this context, including the influence of cell culture conditions and isolation methodologies.

EV enclosure has been primarily studied for naked viruses of the Picornaviridae family, which includes Rhinoviruses (colds), Poliovirus, and Hepatitis A virus (HAV). In Chapters 5-7, we experimentally investigated how naked virus infections influence EV heterogeneity using Encephalomyocarditis virus (EMCV) as a model. In Chapter 5, we examined whether EMCV infection leads to the release of different types of virus-containing and non-containing EVs over time. To study this, we used a laboratory-developed technique called high-resolution flow cytometry, which allows the analysis and sorting of individual EVs. We show that EVs secreted by EMCV-infected cells differ in size, light scattering, molecular composition, and function. Some EVs are more effective at transmitting infection to new cells than others.

In Chapter 6, we examined whether different types of EVs are formed via different routes. The presence of the autophagy-related protein LC3 in EVs from infected cells suggested the involvement of secretory autophagy. We show that secretory autophagy plays a key role in the formation and release of virus-containing EVs and that a viral protein called the 'Leader' is a major factor in this process by suppressing degradative autophagy and stimulating the secretory route.

In Chapter 7, we performed an extensive proteomics analysis of the protein composition of EVs from EMCV-infected cells. We compared proteins in EVs from infected and non-infected cells and investigated the role of the Leader protein using a mutant virus. This analysis provided strong evidence that secretory autophagy is involved in EV formation. It also showed that both the virus and host antiviral defense mechanisms drive the incorporation of specific proteins into EVs, leading to hypotheses about the pro- and antiviral functions of these EVs at different stages of infection.

Heterogeneity in virus-containing EV populations in blood plasma
For most naked viruses, EV enclosure has been demonstrated in cell culture. For HAV and Hepatitis E virus (HEV), virus particles in EVs have also been found in the blood of infected patients. HEV, like HAV, replicates in the liver and can cause hepatitis. Studying virus particles in EVs from human blood is difficult due to the complex composition of plasma, which contains many components (like LDL and HDL) that share physical properties with EVs. In Chapter 8, using various separation techniques, we investigated whether HEV occurs in different EV populations in the blood of infected patients. Preliminary results indicate that HEV is indeed present in multiple types of EVs.

Conclusion
Our findings support the hypothesis that the heterogeneity in molecular composition of EVs is linked to their diverse functions. These data emphasize the importance of mapping the composition of EV populations before their functions can be properly studied.