Mechanisms of uptake of mesenchymal stromal cell membrane particles

Abstract

Background & Aim Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. Even so, culture-expanded MSC are large and get trapped in the pulmonary capillaries after intravenous infusion, where they have a short survival time. To steer cellular immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP) which interact with immune cells and possess immune regulatory properties. However, the intracellular trafficking pathway of MP in recipient cells remains unclear. Methods, Results & Conclusion Here, we characterized MP uptake and internalization by macrophages and endothelial cells. For that, different endocytotic uptake mechanisms of MP, such as phagocytosis, macropinocytosis, dynamin-, clathrin- and caveolin-mediated endocytosis were investigated in human THP-1 macrophages and human umbilical vein endothelial cells (HUVEC) by flow cytometry and confocal microscopy. We demonstrated that MP uptake by macrophages and HUVEC is dose, time, size, and energy-dependent. Also, MP bind to cells through cell-surface receptors, such as heparan sulfate proteoglycans (HSPGs), and are subsequently be internalized. This recipient cell-MP interaction is partially inhibited in the presence of heparin, a soluble analogue of HSPGs. The polarization of M1 and M2 macrophages was achieved by stimulation with LPS and IFN-γ (proinflammatory condition) or IL-4 and IL-13 (anti-inflammatory condition), respectively. Thus, M2 cells exhibited greater endocytic capacity than M1 cells, suggesting that MP may act in particular on regulatory and regenerative macrophages. Under inflammatory stimuli, HUVEC displayed a similar capacity to MP internalization as under control conditions. Moreover, specific inhibitors for endocytotic pathways revealed that MP internalization by macrophages and HUVEC depends on dynamin- and clathrin-mediated endocytosis, but does not involve caveolae-mediated endocytosis. The actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis, are also required in MP uptake by both macrophages and HUVEC. Taken together, these results suggest that MP enter macrophages and HUVEC via a variety of mechanisms, including dynamin- and clathrin-dependent endocytosis, caveolae-independent endocytosis, macropinocytosis, and phagocytosis. Identification of mechanisms involved in MP internalization under different conditions allows specific modulation of MP delivery to target cells.