[Effects of Toxoplasma gondii type I/II rhoptry protein 16 on the polarization and inflammatory response of mouse alveolar macrophages].

Q3 Medicine
J Li, T Dang, Z Zhao
{"title":"[Effects of <i>Toxoplasma gondii</i> type I/II rhoptry protein 16 on the polarization and inflammatory response of mouse alveolar macrophages].","authors":"J Li, T Dang, Z Zhao","doi":"10.16250/j.32.1915.2024199","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>To investigate the effects of <i>Toxoplasma gondii</i> type I and II rhoptry 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 <i>T. gondii</i> infection in host cells and the clinical diagnosis and treatment of pulmonary toxoplasmosis.</p><p><strong>Methods: </strong>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 <i>T. gondii</i> type I and II ROP16 served as the type I and II ROP16 overexpression groups. Following puromycin selection, stably transfected cells that overexpressed type I and II ROP16 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 <i>ROP16</i>, <i>iNOS</i>, <i>IL-1β</i>, <i>IL-4</i>, <i>IL-12</i>, <i>IL-18</i>, <i>Arg-1</i>, <i>IL-10</i>, <i>IL-6</i>, tumor necrosis factor (<i>TNF</i>)-<i>α</i> and transforming growth factor (<i>TGF</i>)-<i>β</i> were detected in mouse alveolar macrophages using RT-qPCR assay.</p><p><strong>Results: </strong>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 (<i>F</i> = 35.30, <i>P</i> < 0.01), and the relative <i>ROP16 mRNA</i> 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 (<i>F</i> = 811.00, <i>P</i> < 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 <i>P</i> 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 (<i>F</i> = 124.70, 82.40, 79.82, 919.40, 84.74, 39.85, 2 354.00 and 65.96, all <i>P</i> 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 <i>P</i> 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 <i>P</i> values < 0.01). RT-qPCR assay detected significant differences among the four groups in terms of <i>iNOS</i>, <i>IL-1β</i>, <i>IL-4</i>, <i>IL-12</i>, <i>IL-18</i>, <i>Arg-1</i>, <i>IL-10</i>, <i>IL-6</i>, <i>TNF-</i>α, and <i>TGF-β mRNA</i> expression (<i>F</i> = 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 <i>P</i> values < 0.05), and the <i>Arg-1</i>, <i>IL-4</i>, <i>IL-10</i>, and <i>TGF-β mRNA</i> expression was significantly higher in the type I ROP16 overexpression group than in the blank control group (all <i>P</i> values < 0.001), while the <i>iNOS</i>, <i>IL-1β</i>, <i>IL-12</i>, <i>IL-18</i>, <i>IL-6</i>, and <i>TNF-α mRNA</i> expression was significantly higher in the type II ROP16 overexpression group than in the blank control group (all <i>P</i> values < 0.001).</p><p><strong>Conclusions: </strong><i>T. gondii</i> 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 <i>T. gondii</i> type I and II ROP16 may activate NLRP3, and mediate the activation of ASC, caspase-1 and IL-1β to promote inflammatory responses.</p>","PeriodicalId":38874,"journal":{"name":"中国血吸虫病防治杂志","volume":"37 2","pages":"127-135"},"PeriodicalIF":0.0000,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"中国血吸虫病防治杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.16250/j.32.1915.2024199","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Medicine","Score":null,"Total":0}
引用次数: 0

Abstract

Objective: To investigate the effects of Toxoplasma gondii type I and II rhoptry 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 type I and II ROP16 served as the type I and II ROP16 overexpression groups. Following puromycin selection, stably transfected cells that overexpressed type I and II ROP16 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.

[刚地弓形虫I/II型弓形虫蛋白16对小鼠肺泡巨噬细胞极化和炎症反应的影响]。
目的:研究弓形虫I型和II型虫体蛋白16 (ROP16)对小鼠肺泡巨噬细胞极化和炎症反应的影响,为揭示弓形虫感染宿主细胞后的免疫调节机制以及肺弓形虫病的临床诊断和治疗提供科学依据。方法:小鼠肺泡巨噬细胞作为空白对照,转染空慢病毒表达载体的小鼠肺泡巨噬细胞作为阴性对照,转染过表达弓形虫I型和II型ROP16的慢病毒载体的小鼠肺泡巨噬细胞作为1型和II型ROP16过表达组。选择嘌呤霉素后,生成稳定转染过表达I型和II型ROP16的细胞,在荧光显微镜下观察绿色荧光表达,并通过PCR、Western blotting和实时定量反转录PCR (RT-qPCR)检测进行验证。采用Western blotting法检测小鼠肺泡巨噬细胞中ROP16、诱导型一氧化氮合酶(iNOS)、精氨酸酶(Arg)-1、甘露糖受体(CD206)、分化集群86 (CD86)、nod样受体热蛋白结构域相关蛋白3 (NLRP3)、caspase-1、含有CARD的凋亡相关斑点样蛋白(ASC)和白细胞介素(IL)-1β蛋白的mRNA表达,以及ROP16、iNOS、IL-1β、IL-4、IL-12、IL-18、Arg-1、IL-10、IL-6、IL-1、IL-1β和IL-1β的mRNA表达水平。采用RT-qPCR法检测小鼠肺泡巨噬细胞中肿瘤坏死因子(TNF)-α和转化生长因子(TGF)-β的表达。结果:荧光显微镜显示,在i型和II型ROP16过表达组和阴性对照组,90%的小鼠肺泡巨噬细胞产生绿色荧光信号。相对ROP16蛋白表达为1.000±0.000,1.003±0.020,1.349±0.055,1.376±0.080空白对照组小鼠肺泡巨噬细胞,负对照组和类型如果IIROP16超表达组(F = 35.30, P < 0.01),和相对ROP16 mRNA表达为1.007±0.172,2.030±0.356,409.579±75.960,413.581±27.712和1在空白对照组,负对照组,和我和II型ROP16超表达组(F = 811.00, P < 0.01)。IIROP16过表达组和IIROP16过表达组ROP16蛋白和mRNA水平均显著高于空白对照组(P值均< 0.01)。免疫印迹分析四组中发现显著差异伊诺,Arg-1, CD86, CD206, NLRP3, caspase-1, ASC, il - 1β蛋白表达(F = 124.70, 82.40, 79.82, 919.40, 84.74, 39.85, 354.00和65.96 2,所有P值< 0.05),Arg-1的表达,CD206, NLRP3, caspase-1, ASC,和il - 1β蛋白显著高于在I型ROP16超表达组比空白对照组(所有P值< 0.001),而伊诺的表达,CD86,II型ROP16过表达组NLRP3、caspase-1、ASC、IL-1β蛋白含量显著高于空白对照组(P值均< 0.01)。RT-qPCR化验发现显著差异在四组伊诺,il - 1β,il - 4、il - 12,地震,Arg-1, il - 10、il - 6、TNF -α,TGF -βmRNA表达(F = 407.00, 528.00, 833.10, 267.90, 989.80, 161.80, 461.10, 529.00 5, 849.60和8 836.00,所有P值< 0.05),和Arg-1, il - 4、il - 10,和TGF -βmRNA表达明显高于在I型ROP16超表达组比空白对照组(所有P值< 0.001),进气阀打开,il - 1β,白介素,地震,II型ROP16过表达组IL-6、TNF-α mRNA表达显著高于空白对照组(P值均< 0.001)。结论:弓形虫IROP16型可诱导小鼠肺泡巨噬细胞m2显性表型,II型ROP16型可诱导小鼠肺泡巨噬细胞m1显性表型。弓形虫I型和II型ROP16均可激活NLRP3,并介导ASC、caspase-1和IL-1β的激活,从而促进炎症反应。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
中国血吸虫病防治杂志
中国血吸虫病防治杂志 Medicine-Medicine (all)
CiteScore
1.30
自引率
0.00%
发文量
7021
期刊介绍: Chinese Journal of Schistosomiasis Control (ISSN: 1005-6661, CN: 32-1374/R), founded in 1989, is a technical and scientific journal under the supervision of Jiangsu Provincial Health Commission and organised by Jiangsu Institute of Schistosomiasis Control. It is a scientific and technical journal under the supervision of Jiangsu Provincial Health Commission and sponsored by Jiangsu Institute of Schistosomiasis Prevention and Control. The journal carries out the policy of prevention-oriented, control-oriented, nationwide and grassroots, adheres to the tenet of scientific research service for the prevention and treatment of schistosomiasis and other parasitic diseases, and mainly publishes academic papers reflecting the latest achievements and dynamics of prevention and treatment of schistosomiasis and other parasitic diseases, scientific research and management, etc. The main columns are Guest Contributions, Experts‘ Commentary, Experts’ Perspectives, Experts' Forums, Theses, Prevention and Treatment Research, Experimental Research, The main columns include Guest Contributions, Expert Commentaries, Expert Perspectives, Expert Forums, Treatises, Prevention and Control Studies, Experimental Studies, Clinical Studies, Prevention and Control Experiences, Prevention and Control Management, Reviews, Case Reports, and Information, etc. The journal is a useful reference material for the professional and technical personnel of schistosomiasis and parasitic disease prevention and control research, management workers, and teachers and students of medical schools.    The journal is now included in important domestic databases, such as Chinese Core List (8th edition), China Science Citation Database (Core Edition), China Science and Technology Core Journals (Statistical Source Journals), and is also included in MEDLINE/PubMed, Scopus, EBSCO, Chemical Abstract, Embase, Zoological Record, JSTChina, Ulrichsweb, Western Pacific Region Index Medicus, CABI and other international authoritative databases.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信