Effects of Toxoplasma gondii type I/II rhoptry protein 16 on the polarization and inflammatory response of mouse alveolar macrophages
10.16250/j.32.1915.2024199
- VernacularTitle:刚地弓形虫Ⅰ/Ⅱ型棒状体蛋白16对小鼠肺泡巨噬细胞极化及炎性反应的影响
- Author:
Jiaming LI
1
,
2
,
3
;
Tiantian DANG
1
,
2
,
3
;
Zhijun ZHAO
1
,
2
,
3
Author Information
1. Laboratory Medical Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, China
2. Ningxia Hui Autonomous Region Key Laboratory of Clinical and Pathogenic Microbiology, Yinchuan, Ningxia 750004, China
3. Ningxia Hui Autonomous Region Clinical Research Center for Laboratory Medicine, Yinchuan, Ningxia 750004, China
- Publication Type:Journal Article
- Keywords:
Toxoplasma gondii;
Rhoptry protein 16;
Mouse alveolar macrophage;
Polarization;
Inflammation
- From:
Chinese Journal of Schistosomiasis Control
2025;37(2):127-135
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of Toxoplasma gondii type Iand 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. MethodsMouse 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 type I and II ROP16 served as the type I and II ROP16 overexpression groups. Following puromycin selection, stably transfected cells that overexpressed type Iand 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 type Iand 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 type Iand 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 type Iand II ROP16 overexpression groups (F = 811.00, P < 0.01). The ROP16 expression was significantly higher in the type Iand 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 type I ROP16 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 II ROP16 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 type I ROP16 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 type II ROP16 overexpression group than in the blank control group (all P values < 0.001). Conclusions T. gondii type IROP16 may induce M2-dominant phenotypes of mouse alveolar macrophages, and type II ROP16 may induce M1-dominant phenotypes of mouse alveolar macrophages. Both T. gondii type I and II ROP16 may activate NLRP3, and mediate the activation of ASC, caspase-1 and IL-1β to promote inflammatory responses.
