Nitric Oxide Production and Apoptosis Induction in T Cells through Stimulation of Mesenchymal Stem Cells with Zymosan

Abstract

Background & Aims: Mesenchymal stem cells (MSCs) are non-hematopoietic multipotant cells, which multiply through attaching to culture plates. Toll-like receptors (TLRs) are inherent immune sensors and regulators of immunomodulatory activities of MSCs. The aim of this study was to investigate the effects of zymosan on stem cell polarization into anti-inflammatory phenotypes through the production of nitric oxide and apoptosis induction in activated T cells. Methods: In this study, MSCs were isolated from mice femur and tibia bone marrow and incubated for 24 hours. Isolation of MSCs with a tendency to stick to the flask was performed during the renewing of the medium. MSCs were grown to 70 percentage of confluence in the medium, and then, they were stimulated with TLR2 agonist (5 and 25 µg/ml doses) and incubated with 5% carbon dioxide for 1 and 12 hours in 37 ºC. Supernatant medium was collected to measure nitric oxide production using Graise test. Percentage of apoptosis in activated T cells was measured using anti-CD3PE, acridin orange/propidium iodide (PI/AO) staining and flow cytometry. Results: We found that the rate of apoptosis in activated T cells increased significantly in cells incubated with a 5 µg/ml dose of zymosan for 1 hour in comparison with the control group (P < 0.05). Moreover, a significant increase was observed in nitric oxide production of cells treated with the same dose for 12 hours (P < 0.05). Conclusion: Based on these results, we can conclude that different doses, TLR agonist type, and duration of incubation impact nitric oxide production and apoptosis rate in activated T cells.