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

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