{"title":"[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]。","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":"{\"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}","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}
[Effects of Toxoplasma gondii type I/II rhoptry protein 16 on the polarization and inflammatory response of mouse alveolar macrophages].
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.
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.
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