Innovative approaches to study exosome biogenesis and uptake: Insights into Mannose Receptor-mediated uptake and immunomodulatory effects on Dendritic Cells

Exosomes are gaining recognition as important mediators of intercellular communication, and researcher are focused on their potential clinical applications. However, challenges in accurately isolating exosomes from other types of Extracellular Vesicles (EVs) have hindered both their functional studies and clinical utility. Moreover, many aspects related to exosome biogenesis, interaction with recipient cells, and selective uptake mechanisms remain poorly understood. The aim of this study was to investigate the mechanisms of exosomes internalization and their and immunomodulatory effects on immune cells. To distinguish exosomes from the other EVs, we used the Bodipy FL C16, a fluorescent palmitic acid analogue, that enabled us to identify a discrete subpopulation of small exosomes.The Bodipy C16 is incorporated in the fluorescent phospholipids into specific intracellular organelles, culminating in their integration into the exosomal membrane. The characterization of Bodipy exosomes sheds light on their enrichment in tetraspanin markers, notably CD63 and CD81, along with a minor proportion of CD9. The authentication of the exosomal nature of Bodipy vesicles is achieved through nanoFACS sorting and electron microscopy. As awareness of the pivotal role of exosomes, particularly in immune modulation, continues to grow, the question of their selective or non-selective uptake by recipient cells becomes a central focus. Recent studies have demonstrated that exosomes are particularly enriched in high mannose glycans. Therefore, we investigated exosome internalization by antigen-presenting cells, specifically immature dendritic cells (iDCs) characterized by their expression of the mannose receptor (MR). Hypoxic conditions, which are prevalent in the tumor microenvironment, can induce changes in glycosylation patterns. Thus, we aimed to evaluate the specific uptake mediated by the MR of exosomes secreted under normoxic or hypoxic conditions. This study not only explores the uptake of Bodipy exosomes by iDCs but also elucidates their influence on these cells through fluorescence quantification. The results reveal that exosomes are selectively internalized via the MR by iDCs, prompting significant phenotypic transformations within the DCs, thereby shedding light on the profound immunomodulatory capabilities of exosomes within the innate immune system. This findings shed light on the immunomodulatory properties of exosomes in the innate immune system. However, further research is required to fully understand the underlying mechanisms and utilize the therapeutic potential of these results.