Figure 1.

Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM CellTrace™ Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10⁵/well) were co-cultured with ApoMSC (4 × 10⁵/well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10⁵/well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2^high, n=3 (E), IDO^high, n=4 (F) and PD-L1^high, n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ FarRed (APC⁺) while monocytes were labeled with CellTrace™ Violet (V450⁺). APC⁺ monocytes were regarded as efferocytosis⁺ and APC⁻ monocytes were regarded as efferocytosis⁻. (L) Bar charts showing the difference of COX2^high, IDO^high and PD-L1^high populations between the efferocytosis⁺ and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).