Figure 5

S-BMMSCs show up-regulated immunomodulatory properties through nitric oxide (NO) production. (A) NO levels in the supernatant of S-BMMSC and BMMSC culture were significantly higher in the INF-γ/IL-1β treated S-BMMSC group than in BMMSCs. (B-C) S-BMMSCs showed a significant reduction in the cell viability of activated SP cells compared to the cells cultured without BMMSCs (SP cell) and with BMMSCs (B). Both BMMSCs and S-BMMSCs showed a significantly increased rate of SP cell apoptosis compared to the SP cell only group but S-BMMSCs could induce higher SP cell apoptosis (C). (D-E) The induction of SP cell apoptosis by BMMSCs or S-BMMSCs was abolished in general NOS inhibitor L-NMMA-treated (D) and iNOS specific inhibitor 1400 W-treated (E) group. (F-H) Activated CD4⁺CD25^- T-cells and S-BMMSCs or BMMSCs were co-cultured in the presence of TGFβ1 and IL-2 with or without NOS inhibitor for three days. The floating cells were stained for CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs). Both BMMSCs and S-BMMSC up-regulated Tregs but S-BMMSCs showed a significant effect in up-regulating Tregs. (F). Interestingly, L-NMMA and 1400 W treatments resulted in an abolishing of S-BMMSC-induced up-regulation of Tregs (G, H). (I) BMMSCs and S-BMMSCs could inhibit Th17 differentiation in vitro. S-BMMSC could inhibit it more effectively. (J, K) L-NMMA (J) or 1400 W (K) could abolish the inhibition of Th17 differentiation by BMMSCs or S-BMMSCs. The results are representative of at least three independent experiments. *P <0.05; **P <0.01; ***P <0.001. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; iNOS, inducible nitric oxide synthase; L-NMMA, L-NG-monomethyl-arginine; NOS, nitric oxide synthase; S-BMMSCs, BMMSCs in suspension; SD, standard deviation; SP, spleen; Tregs, regulatory T cells.