ABSTRACT
Objective To investigate the in vitro effect of alginate (Alg)/glycol chitosan (GC) double-network hydrogel on immune function of splenic regulatory T cells (Treg) of mice. Methods Single network hydrogel of Alg or GC and double-network (DN) gel material (Alg/GC) were prepared. Splenic CD4+CD25+Treg of BABL/c mice were isolated and purified by Magnetic Activated Cell Sorting (MACS) kit and further stimulated with different hydrogels for 24 hours and 48 hours with or without polymyxin B (PMB) exposure. Cells treated with Alg, or GC, or DN were divided into Alg group, GC group, DN group, respectively. Cells without hydrogens were served as control group. Expressions of the forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T cell-associated antigen 4 (CTLA-4) on CD4+CD25+Treg were measured by flow cytometry. Their expression levels would be used to determine the conditions for further experiments. The CFSE kit was applied for assessing the proliferative activity of effector T cells (Teffs) after co-cultured with CD4+CD25+Treg. Enzyme linked immunosorbent assay (ELISA) was used to determine the cytokine levels including interleukin (IL)-10 and transforming growth factor (TGF)-β, IL-2, IL-4, and interferon (IFN)-γ. Results With or without PMB exposure, the expression of CTLA-4 and Foxp3 showed no significant difference under the stimulation of various hydrogels (P>0.05), including Alg, GC, and DN. Therefore, for further studies, cells were stimulated by different hydrogens in the presence of PMB for 24 hours. Cells without hydrogen simulation were served as control. Compared with the control group [(47.73±5.35)%], CTLA-4 expression was obviously up-regulated in both GC [(95.99±2.79)%] and DN [(92.21±2.97)%] hydrogel groups (P<0.05). In addition, TGF-β levels were markedly elevated in CD4+CD25+Treg stimulated with GC [(957.06±138.70)pg/ml] or DN [(905.03±73.04)pg/ml], compared with the level in the cells in the control group [(558.75±48.58)pg/ml, P<0.05]. However, no difference was detected in the expression of Foxp3 among different groups. In comparison with control group [proliferation (42.04±1.35)%, IL-4/IFN-γ (1.45±0.27)], Treg cells that were stimulated with GC or DN showed enhanced inhibitory effect on Teffs, including suppressed proliferation of Teffs [DN group (37.22±1.39)%, GC group (35.20±2.03)%] and increased ratio of IL-4/IFN-γ of Teffs [DN group (2.30±0.22), GC group (2.57±0.23)] (P<0.05). Conclusion Alg/GC double-network hydrogel can greatly enhance the immunosuppressive response of Treg, which is dominantly seen with GC single network hydrogel.