Abstract: 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). Conclusion　Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP?1?derived macrophages to M2 type in vitro.

Key words: Nippostrongylus brasiliensis, L3 protein, L5 protein, THP?1 cell, Macrophage polarization

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。

关键词: 巴西日圆线虫, L3蛋白, L5蛋白, THP?1细胞, 巨噬细胞极化