巴西日圆线虫虫体蛋白体外诱导THP-1来源巨噬细胞极化的研究

Polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro

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MO型细胞分别采用500 ng/mL脂多糖(LPS)+100 ng/mLγ-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MO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(χ~2=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(χ~2=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(χ~2=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。

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 MO 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(χ~2=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(χ~2=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-12 b(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(χ~2=24 004.33 and 832.50,P <0.001).Higher IL-1(3 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.