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

Hong Xie; Liang Chu; Ling-qin WU; Xing-yu Fan; Pu Wang; Si-yu MA; Dong-xue Zheng; Kun-long LI; Xing-zhi Chen; Xiao-di Yang

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

VernacularTitle:重组旋毛虫半胱氨酸蛋白酶抑制剂体外诱导骨髓来源巨噬细胞极化的研究
Author: Hong XIE ^{1
,

2} ; Liang CHU ³ ; Ling-Qin WU ^{4
,

5} ; Xing-Yu FAN ⁴ ; Pu WANG ⁴ ; Si-Yu MA ⁴ ; Dong-Xue ZHENG ⁴ ; Kun-Long LI ⁴ ; Xing-Zhi CHEN ^{1
,

2} ; Xiao-Di YANG ^{1
,

2}
Author Information

1. Basic Medical College, Bengbu Medical College, Bengbu 233000, China
2. Anhui Key Laboratory of Infection and Immunity, China
3. The Second Affiliated Hospital of Bengbu Medical College, China
4. Anhui Key Laboratory of Infection and Immunity, China
5. The First Affiliated Hospital of Bengbu Medical College, China
Publication Type:Journal Article
Keywords: Trichinella spiralis; Cysteine protease inhibitor; Macrophage; Immune regulation
From: Chinese Journal of Schistosomiasis Control 2020;32(2):181-186
CountryChina
Language:Chinese
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-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). Conclusion rTs-Cys may induce the polarization of BMDMs to antiinflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.