Radical-Generating Activity, Phagocytosis, and Mechanical Properties of Four Phenotypes of Human Macrophages.

Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes-pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics-high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities.

MSCs' conditioned media cytokine and growth factor profiles and their impact on macrophage polarization.

BACKGROUND: There is a growing body of evidence that multipotent mesenchymal stromal cells' (MSCs') remarkable therapeutic potential is attributed not only to their differentiation and regenerative capacity, but also to the paracrine effect, underlying their immunomodulatory properties. MSCs' secretome (i.e., cytokines, growth factors, and extracellular vesicles) is therefore increasingly discussed in the context of their ability to modulate inflammatory response and promote regeneration. There is evidence that 2D or 3D culturing conditions have an impact on the cells' secretome, and here we aimed to compare the secretion of cytokines and growth factors in human MSCs from different sources cultured in 2D and 3D conditions and assess their effect on human macrophages polarization in vitro. METHODS: MSCs were derived from human adipose tissue, bone marrow, gingiva, placenta, and umbilical cord, cultured as monolayers or as cell spheroids. Their cytokine profiles were analyzed, and data standardization was carried out using a z-score. Human peripheral blood mononuclear cells-derived macrophages were then treated with umbilical cord-derived MSCs' conditioned media and their effect on macrophages polarization was assessed. RESULTS: Our findings suggest that umbilical cord-derived MSCs' conditioned media demonstrated the highest cytokine and growth factor levels and despite mostly pro-inflammatory cytokine profile were able to promote anti-inflammatory macrophage polarization. CONCLUSIONS: Umbilical cord-derived MSCs' conditioned media hold great potential for therapeutic use, demonstrating significant anti-inflammatory effect on human macrophages.

Redox-Activation of Neutrophils Induced by Pericardium Scaffolds.

Implantation of scaffolds causes a local inflammatory response whereby the early recruitment of neutrophils is of great importance not only for fighting the infection, but also for facilitating effective regeneration. We used luminol-dependent chemiluminescence, flow cytometry, ELISA, and confocal microscopy to assess the responses of neutrophils after the exposure to the scaffold-decellularized bovine pericardium (collagen type I) crosslinked with genipin (DBPG). We demonstrated that DBPG activated neutrophils in whole blood causing respiratory burst, myeloperoxidase (MPO) secretion, and formation of neutrophil extracellular trap-like structures (NETs). In addition, we studied platelets, another important player of the immediate immune host response. We found that platelets triggered redox-activation of isolated neutrophils by the pericardium scaffold, and likely participate in the NETs formation. Free radicals generated by neutrophils and hypochlorous acid produced by MPO are potent oxidizing agents which can oxidatively degrade biological structures. Understanding the mechanisms and consequences of redox activation of neutrophils by pericardium scaffolds is important for the development of new approaches to increase the efficiency of tissue regeneration.