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-β (TGF-β) 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-β (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL-10 and TGF-β levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF-β 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.
［摘要］ 目的 探讨重组旋毛虫半胱氨酸蛋白酶抑制 (rTs-Cys) 对体外诱导骨髓来源巨噬细胞 (BMDMs) 极化的调控作用。方法 获取BMDMs并培养至条件培养基中, 7 d 后获取成熟BMDMs。将此BMDMs分为阴性对照组 (A组)、阳性对照组 (B组)、单独重组蛋白组 (C组) 和蛋白共培养组 (D组), A组细胞给予 10 ng/mL γ-干扰素 (IFN-γ) 和 100 ng/mL 脂多糖 (LPS) 刺激, B组细胞给予 10 ng/mL 白细胞介素 (IL) -4和 10 ng/mL IL-10刺激, C组细胞给予 1 μg/mL rTs-Cys干预, D组细胞给予 1 μg/mL rTs-Cys、10 ng/mL IFN-γ及 100 ng/mL LPS刺激。干预 24 h 后, 收集细胞及培养上清, 采用流式细胞术检测各组细胞中F4/80+、CD11b+、CD206+ 和CD11c+ 细胞比例, 采用酶联免疫吸附试验 (ELISA) 检测细胞培养上清中IL-6、肿瘤坏死因子-α (TNF-α)、IL-10及转化生长因子-β (TGF-β) 水平, 采用免疫荧光染色鉴定CD86+、CD206+ 表型。结果 流式细胞术检测结果显示, 4 组F4/80+ CD11b+ CD11c+ 细胞比例差异具有统计学意义 (F = 46.184, P < 0.001), C组及D 组F4/80+ CD11b+ CD11c+ 细胞比例均较A组显著降低 (P 均< 0.001); 4 组 F4/80+ CD11b+ CD206+ 细胞比例差异具有统计学意义 (F = 11.032, P < 0.01), C 组及 D 组 F4/80+ CD11b+ CD206+ 细胞比例均较A组显著升高 (P 均< 0.01)。免疫荧光染色显示, C组及D组CD206+ 表达水平较A组显著上调, 两组CD86+ 表达水平较 A 组下降。ELISA 检测结果显示, 4 组细胞培养上清液中 IL-6 (F = 3.950, P < 0.001) 和 TNF-α 水平 (F = 205.827, P < 0.001) 差异均有统计学意义; C 组及 D 组 IL-6 和 TNF-α 水平均较A组显著下降 (P 均< 0.05)。4 组细胞培养上清液中 IL-10 (F =8.274, P < 0.001) 和 TGF-β 水平 (F = 13.559, P < 0.01) 差异均有统计学意义; C 组 IL-10、TGF-β 水平较A组显著增加 (P 均< 0.01); D 组 TGF-β 水平较A组显著增加 (P < 0.05), 但 IL-10 水平较 A 组无明显变化 (P > 0.05)。结论 rTs-Cys 体外能诱导 BMDMs 向抗炎特性的M2方向极化, 并抑制 M1 型巨噬细胞活化。.
Keywords: Cysteine protease inhibitor; Immune regulation; Macrophage; Trichinella spiralis.