Observation capabilities and opportunities

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Efferocytosis of viable versus heat-inactivated MSC induces human monocytes to distinct immunosuppressive phenotypes

Abstract Background Immunomodulation by mesenchymal stromal cells (MSCs) can occur through trophic factor mechanisms, however, intravenously infused MSCs are rapidly cleared from the body yet a potent immunotherapeutic response is still observed. Recent work suggests that monocytes contribute to the clearance of MSCs via efferocytosis, the body’s natural mechanism for clearing dead and dying cells in a non-inflammatory manner. This begs the questions of how variations in MSC quality affect monocyte phenotype and if viable MSCs are even needed to elicit an immunosuppressive response. Methods Herein, we sought to dissect MSC’s trophic mechanism from their efferocytic mechanisms and determine if the viability of MSCs prior to efferocytosis influences the resultant phenotype of monocytes. We cultured viable or heat-inactivated human umbilical cord MSCs with human peripheral blood mononuclear cells for 24 h and observed changes in monocyte surface marker expression and secretion profile. To isolate the effect of efferocytosis from MSC trophic factors, we used cell separation techniques to remove non-efferocytosed MSCs before challenging monocytes to suppress T-cells or respond to inflammatory stimuli. For all experiments, viable and heat-inactivated efferocytic-licensing of monocytes were compared to non-efferocytic-licensing control. Results We found that monocytes efferocytose viable and heat-inactivated MSCs equally, but only viable MSC-licensed monocytes suppress activated T-cells and suppression occurred even after depletion of residual MSCs. This provides direct evidence that monocytes that efferocytose viable MSCs are immunosuppressive. Further characterization of monocytes after efferocytosis showed that uptake of viable-but not heat inactivated-MSC resulted in monocytes secreting IL-10 and producing kynurenine. When monocytes were challenged with LPS, IL-2, and IFN-γ to simulate sepsis, monocytes that had efferocytosed viable MSC had higher levels of IDO while monocytes that efferocytosed heat inactivated-MSCs produced the lowest levels of TNF-α. Conclusion Collectively, these studies show that the quality of MSCs efferocytosed by monocytes polarize monocytes toward distinctive immunosuppressive phenotypes and highlights the need to tailor MSC therapies for specific indications

Additional file 1 of Efferocytosis of viable versus heat-inactivated MSC induces human monocytes to distinct immunosuppressive phenotypes

Additional file 1. Figure S1: Heat-inactivation sets MSCs on a path toward apoptosis (related to Figure 1). Figure S2: Residual Non-phagocytosed viable MSC spheroids were transferred from 24hr efferocytic-licensing to T-cell activation plates (related to Figure 2). Figure S3: MSC depletion efficiency and dose-dependent immunomodulatory potency of healthy adherent MSC toward activated T-cells (related to Figure 3). Figure S4: Gating strategy for surface marker analysis of monocytes following MSC efferocytosis. Figure S5: CD206 expression of CD14+ monocytes without and with viable MSC efferocytic-licensing (related to Figure 4). Figure S6: Monocyte isolation is efficient and isolated monocytes phagocytose viable and HI-MSCs in the absence of Tcells (related to Figure 1)

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