Abstract: Objective　To investigate the effects of Toxoplasma gondii typeⅠand Ⅱrhoptry protein 16 (ROP16) on the polarization and inflammatory response of mouse alveolar macrophages, so as to provide the scientific evidence for unveiling the immunoregulatory mechanisms following T. gondii infection in host cells and the clinical diagnosis and treatment of pulmonary toxoplasmosis. Methods　Mouse alveolar macrophages served as blank controls, and mouse alveolar macrophages transfected with the empty lentiviral expression vector served as negative controls, and mouse alveolar macrophages transfected with lentiviral vectors overexpressing T. gondii typeⅠand Ⅱ ROP16 served as the typeⅠand ⅡROP16 overexpression groups. Following puromycin selection, stably transfected cells that overexpressed typeⅠand ⅡROP16 were generated, observed for green fluorescence expression under a fluorescence microscope and verified using PCR, Western blotting and real⁃time quantitative reverse transcription PCR (RT⁃qPCR) assays. The expression of ROP16, inducible nitric oxide synthase (iNOS), arginase (Arg)⁃1, mannose receptor (CD206), cluster of differentiation 86 (CD86), NOD⁃like receptor thermal protein domain associated protein 3 (NLRP3), caspase⁃1, apoptosis⁃associated speck⁃like protein containing a CARD (ASC), and interleukin (IL)⁃1β proteins was determined in mouse alveolar macrophages using Western blotting assay, and the mRNA levels of ROP16, iNOS, IL⁃1β, IL⁃4, IL⁃12, IL⁃18, Arg⁃1, IL⁃10, IL⁃6, tumor necrosis factor (TNF)⁃α and transforming growth factor (TGF)⁃β were detected in mouse alveolar macrophages using RT⁃qPCR assay. Results　Fluorescence microscopy showed 90% of mouse alveolar macrophages producing green fluorescent signals in the typeⅠand Ⅱ ROP16 overexpression groups and the negative control group. The relative ROP16 protein expression was  1.000 ± 0.000, 1.003 ± 0.020, 1.349 ± 0.055, and 1.376 ± 0.080 in mouse alveolar macrophages in the blank control group, negative control group, and typeⅠand ⅡROP16 overexpression groups (F = 35.30, P < 0.01), and the relative ROP16 mRNA expression was 1.007 ± 0.172, 2.030 ± 0.356, 1 409.579 ± 75.960, and 1 413.581 ± 27.712 in the blank control group, negative control group, and typeⅠand Ⅱ ROP16 overexpression groups (F = 811.00, P < 0.01). The ROP16 expression was significantly higher in the typeⅠand ⅡROP16 overexpression groups than in the blank control group at both protein and mRNA levels (all P value < 0.01). Western blotting assay detected significant differences among the four groups in terms of iNOS, Arg⁃1, CD86, CD206, NLRP3, caspase⁃1, ASC, and IL⁃1β protein expression (F = 124.70, 82.40, 79.82, 919.40, 84.74, 39.85, 2 354.00 and 65.96, all P values < 0.05), and the expression of Arg⁃1, CD206, NLRP3, caspase⁃1, ASC, and IL⁃1β proteins was significantly higher in the typeⅠROP16 overexpression group than in the blank control group (all P values < 0.001), while the expression of iNOS, CD86, NLRP3, caspase⁃1, ASC, and IL⁃1β proteins was significantly higher in the type ⅡROP16 overexpression group than in the blank control group (all P values < 0.01). RT⁃qPCR assay detected significant differences among the four groups in terms of iNOS, IL⁃1β, IL⁃4, IL⁃12, IL⁃18, Arg⁃1, IL⁃10, IL⁃6, TNF⁃α, and TGF⁃β mRNA expression (F = 407.00, 1 528.00, 833.10, 267.90, 989.80, 161.80, 461.10, 5 529.00, 849.60 and 8 836.00, all P values < 0.05), and the Arg⁃1, IL⁃4, IL⁃10, and TGF⁃β mRNA expression was significantly higher in the typeⅠROP16 overexpression group than in the blank control group (all P values < 0.001), while the iNOS, IL⁃1β, IL⁃12, IL⁃18, IL⁃6, and TNF⁃α mRNA expression was significantly higher in the typeⅡROP16 overexpression group than in the blank control group (all P values < 0.001). Conclusions　T. gondii typeⅠROP16 may induce M2⁃dominant phenotypes of mouse alveolar macrophages, and typeⅡROP16 may induce M1⁃dominant phenotypes of mouse alveolar macrophages. Both T. gondii typeⅠand ⅡROP16 may activate NLRP3, and mediate the activation of ASC, caspase⁃1 and IL⁃1β to promote inflammatory responses.

Key words: Toxoplasma gondii, Rhoptry protein 16, Mouse alveolar macrophage, Polarization, Inflammation

摘要： 目的　分析刚地弓形虫Ⅰ、Ⅱ型棒状体蛋白16（rhoptry protein 16，ROP16）对小鼠肺泡巨噬细胞极化状态及炎性反应的影响，为揭示弓形虫感染宿主细胞后机体免疫调节机制及肺弓形虫病临床诊治提供科学依据。方法　以小鼠肺泡巨噬细胞为空白对照组，转染空载体慢病毒的小鼠肺泡巨噬细胞为阴性对照组，转染Ⅰ、Ⅱ型ROP16过表达慢病毒的小鼠肺泡巨噬细胞为Ⅰ、Ⅱ型 ROP16过表达组。经嘌呤霉素筛选获得稳定表达Ⅰ、Ⅱ型ROP16的过表达稳转细胞株，在荧光显微镜下观察细胞绿色荧光表达，并通过PCR、Western blotting以及实时荧光定量PCR（quantitative real⁃time PCR，RT⁃qPCR）进行验证。利用 Western blotting检测各组细胞ROP16、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、精氨酸酶（arginase，Arg）⁃1、甘露糖受体（mannose receptor，CD206）、分化簇 86（cluster of differentiation 86，CD86）、抗 NOD样受体热蛋白结构域相关蛋白3（NOD⁃like receptor thermal protein domain associated protein 3，NLRP3）、半胱天冬酶（caspase）⁃1、凋亡相关斑点蛋白（apoptosis⁃associated speck⁃like protein containing a CARD，ASC）、白细胞介素（interleukin，IL）⁃1β蛋白表达水平，采用RT⁃qPCR检测各组ROP16、iNOS、IL⁃1β、IL⁃4、IL⁃12、IL⁃18、Arg⁃1、IL⁃10、IL⁃6、肿瘤坏死因子（tumor necrosis factor，TNF）⁃α和转化生长因子（transforming growth factor，TGF）⁃β mRNA水平。结果　弓形虫Ⅰ、Ⅱ型ROP16过表达组及阴性对照组细胞均可观察到90%的细胞表达绿色荧光，空白对照组、阴性对照组及Ⅰ、Ⅱ型ROP16过表达组细胞中ROP16蛋白相对表达量分别为1.000 ± 0.020、1.003 ± 0.020、1.349 ± 0.055、1.376 ± 0.080，ROP16 mRNA相对表达量分别为1.007 ± 0.172、2.030 ± 0.356、1 409.579 ± 75.960、1 413.581 ± 27.712，差异均有统计学意义（F = 35.30、811.00，P 均< 0.01）；两两比较结果显示，Ⅰ、Ⅱ型ROP16过表达组细胞中ROP16蛋白及mRNA表达水平均较空白对照组显著升高（P均 < 0.01）。Western blotting 检测结果显示，4组细胞中iNOS、Arg⁃1、CD86、CD206、NLRP3、caspase⁃1、ASC和IL⁃1β蛋白表达水平差异均有统计学意义（F = 124.70、82.40、79.82、919.40、84.74、39.85、2 354.00、65.96，P均< 0.05）；两两比较结果显示，Ⅰ型ROP16过表达组细胞中Arg⁃1、CD206、NLRP3、caspase⁃1、ASC和IL⁃1β蛋白表达水平均较空白对照组显著上调（P均< 0.001）；Ⅱ型ROP16过表达组细胞中iNOS、CD86、NLRP3、caspase⁃1、ASC和 IL⁃1β蛋白表达水平均较空白对照组显著上调（P均< 0.001）。RT⁃qPCR检测结果显示，4组iNOS、IL⁃1β、IL⁃4、IL⁃12、IL⁃18、Arg⁃1、IL⁃10、IL⁃6、TNF⁃α、TGF⁃β mRNA表达水平差异均有统计学意义（F = 407.00、1 528.00、833.10、267.90、989.80、161.80、461.10、5 529.00、849.60、        8 836.00，P 均< 0.05）；两两比较结果显示，Ⅰ型ROP16过表达组细胞中Arg⁃1、IL⁃4、IL⁃10、TGF⁃β mRNA表达水平均较空白对照组显著上调（P 均< 0.001），Ⅱ型ROP16过表达组细胞中iNOS、IL⁃1β、IL⁃12、IL⁃18、IL⁃6、TNF⁃α mRNA表达水平均较空白对照组显著上调（P均< 0.001）。结论　刚地弓形虫Ⅰ型ROP16蛋白可诱导小鼠肺泡巨噬细胞极化为M2为主的表型、Ⅱ型ROP16蛋白可诱导小鼠肺泡巨噬细胞极化为M1为主的表型；Ⅰ、Ⅱ 型ROP16蛋白均可激活NLRP3，介导ASC、caspase⁃1和IL⁃1β活化，促进炎症反应发生。

关键词: 刚地弓形虫, 棒状体蛋白16, 小鼠肺泡巨噬细胞, 极化, 炎症