Objective: To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections.
Methods: The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro.
Results: Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1β and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-β1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05).
Conclusions: Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.
［摘要］ 目的 分析巴西日圆线虫虫体蛋白体外诱导人单核细胞白血病THP-1细胞来源巨噬细胞的极化方向, 探索机体对巴西日圆线虫感染的免疫应答机制。方法 建立并维持巴西日圆线虫培养的实验室循环, 无菌条件下收集L3和L5虫体, 分别制备虫体蛋白; 建立THP-1细胞体外培养体系, 经500 ng/mL佛波酯 (PMA) 刺激培养后呈贴壁状态的M0型细胞分别采用500 ng/mL脂多糖 (LPS) + 100 ng/mL γ-干扰素 (IFN-γ)、白细胞介素4 (IL-4) + IL-13 (浓度均为100 ng/mL)、L3及L5虫体蛋白 (浓度均为5 mg/mL) 进行刺激, 同时设不加任何刺激的阴性对照组。通过显微镜镜检观察刺激后细胞形态、实时荧光定量PCR (qPCR) 检测M1/M2型巨噬细胞特异性基因mRNA 表达水平、流式细胞术检测巨噬细胞表面标志物及酶联免疫吸附试验 (ELISA) 检测M1/M2型巨噬细胞分泌的细胞因子含量, 观察巴西日圆线虫虫体蛋白体外诱导THP-1来源巨噬细胞的极化方向。结果 THP-1细胞经PMA刺激培养呈贴壁的M0型细胞后, 经LPS + IFN-γ刺激培养后呈特征性M1型极化; 经IL-4 + IL-13刺激培养后呈特征性M2型极化; 经L3和L5蛋白刺激培养后均呈特征性M2型极化。阴性对照组、LPS + IFN-γ刺激组、IL-4 + IL-13刺激组、L3蛋白刺激组、L5蛋白刺激组间M1型巨噬细胞比例差异有统计学意义 (χ2 = 3 721.00, P < 0.001), 其中LPS + IFN-γ刺激组M1型巨噬细胞比例最高; 各组间M2型巨噬细胞比例差异有统计学意义 (χ2 = 105.43, P < 0.001)。各组间C-C基序趋化因子配体2 (CCL2)、肿瘤坏死因子α (TNF-α)、IL-12b、过氧化物酶体增殖物激活受体γ (PPARγ)、IL-10、甘露糖受体C型1 (Mrc1) 基因mRNA 表达水平差异均有统计学意义 (F =191.95、129.95、82.89、11.30、9.51、12.35, P 均< 0.001), 各组间CD86、CD206阳性率差异均有统计学意义 (χ2 = 24 004.33、832.50, P 均< 0.001)。LPS + IFN-γ刺激组IL-1β、TNF-α表达水平均显著高于IL-4 + IL-13刺激组、L3蛋白刺激组及L5蛋白刺激组 (P 均< 0.001) ; IL-4 + IL-13刺激组、L3蛋白刺激组和L5蛋白刺激组TGF-β1、IL-10表达水平均显著高于阴性对照组和LPS + IFN-γ刺激组 (P 均< 0.05)。结论 巴西日圆线虫L3和L5虫体蛋白体外均可诱导THP-1来源巨噬细胞向M2型极化。.
Keywords: L3 protein; L5 protein; Macrophage polarization; Nippostrongylus brasiliensis; THP-1 cell.