[Polarization of bone marrow-derived macrophages induced by recombinant Trichinella spiralis cysteine protease inhibitors in vitro].

ABSTRACT

OBJECTIVE: To investigate the regulatory role of recombinant Trichinella spiralis cysteine protease inhibitors (rTs-Cys) in induction of polarization of bone marrow-derived macrophages (BMDMs) in vitro. METHODS: BMDMs were captured and cultured in conditioned medium for 7 days. Then, mature BMDMs were harvested and assigned into four groups. Cells in Group A (negative control) were given 10 ng/mL IFN-γ combined with 100 ng/mL LPS, cells in Group B (positive control) were treated with IL-4 and IL-10 (at 10 ng/mL both), and cells in Group C (recombinant protein alone) were stimulated with 1 µg/mL rTs-Cys, while cells in Group D (protein co-culture) were simultaneously treated with 1 µg/mL rTs-Cys, 10 ng/mL IFN-γ and 100 ng/mL LPS. Cells and culture supernatant were collected 24 hour post-treatment, and the proportions of F4/80+, CD11b+, CD206+ and CD11c+ cells were detected by flow cytometry. The levels of interleukin IL-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 and transforming growth factor-ß (TGF-ß) in the cell culture supernatant were measured by ELISA and the CD86+ and CD206+ phenotypes were identified by immunofluorescent staining. RESULTS: Flow cytometry detected no significant difference in the proportion of F4/80+ CD11b+ CD11c+ cells among the four groups (F = 46.184, P < 0.001), and a lower proportion of F4/80+ CD11b+ CD11c+ cells was seen in groups C and D than in group A (all P values < 0.001). There was a significant difference in the proportion of F4/80+ CD11b+ CD206+ cells among the four groups (F = 11.032, P < 0.001), and a greater proportion of F4/80+ CD11b+ CD206+ cells was seen in groups C and D than in group A (all P values < 0.01). Immunofluorescent staining showed higher CD206+ expression and lower CD86+ expression in groups C and D than in Group A. There were significant differences in the IL-6 and (F = 3.950, P < 0.001) and TNF-α (F = 205.827, P < 0.001) levels in the cell culture supernatants among the four groups, and significantly lower IL-6 and TNF-α levels were measured in groups C and D than in Group A (both P < 0.05). There were significant differences in the IL-10 and (F = 8.274, P < 0.001) and TGF-ß (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL-10 and TGF-ß levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF-ß level was significantly higher in Group D than in Group A (P < 0.05); however, there was no significant difference in the IL-10 level between groups D and A (P > 0.05). CONCLUSIONS: rTs-Cys may induce the polarization of BMDMs to antiinflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.