CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2022 , Vol. 40 >Issue 5: 579 - 586

DOI: https://doi.org/10.12140/j.issn.1000-7423.2022.05.003

ORIGINAL ARTICLES

Effects of ROP16 protein of Toxoplasma gondii on polarization and apoptosis of MH-S cells and their related mechanisms

  • LI Jia-ming ,
  • WANG Yi-xuan ,
  • YANG Ning-ai ,
  • MA Hui-hui ,
  • LAN Min ,
  • LIU Chun-lan ,
  • ZHAO Zhi-jun
Expand
  • 1. School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
    2. Ningxia Key Laboratory of Pathogenic Microbiology, Yinchuan 750004, China
    3. Medical Experiment Center, General Hospital of Ningxia Medical University, Yinchuan 750004, China

Received date: 2022-03-22

  Revised date: 2022-05-04

  Online published: 2022-09-14

Fold

Abstract

Objective To investigate the expression of Toxoplasma gondii rhoptry protein 16 (ROP16) protein in mouse alveolar macrophages (MH-S) and its affect on cell polarization and apoptosis and the mechanisms involved. Methods MH-S cells transfected with ROP16 overexpression lentivirus labeled of green fluorescent were used to construct ROP16-MH-S cell line capable of stable expression of ROP16 (overexpression group). A blank vector lentivirus transfection control group (blank vector group) and no transfection control group (control group) were assigned in parallel. The expression of ROP16 in ROP16-MH-S and its localization within the cells were detected by immunofluorescence 72 h post-transfection. With the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal reference, and the mRNA relative transcription level of pro-inflammatory (M1) factor interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-18, IL-6 and IL-12; apoptosis-suppressing gene B-cell lymphoma/leukemia-2 (Bcl-2); and anti-inflammatory (M2) factors IL-10, transforming growth factor (TGF)-β, pro-apoptotic genes Bcl-2-associated X protein (Bax), cysteine protease 3 (Caspase 3), Caspase 9 in ROP16-MH-S cells was detected by RT-qPCR. The relative expression level of polarized protein arginase 1 (Arg-1), signal transducer and activator of transcription 3 (STAT3), phosphorylation at site 705 (pTyr705)-STAT3, STAT6, pTyr641-STAT6 and apoptosis proteins Caspase 9, Caspase 3, Bcl-2 and Bax in ROP16-MH-S cells were detected by Western-blotting. The apoptosis of ROP16-MH-S cells was detected by flow cytometry. One-way analysis of variance was used for comparison between groups. Results Strong green fluorescence was seen in the blank vector group and overexpression group at 72 h after transfection. Significant red fluorescence was also observed in the nucleus of ROP16-MH-S cells and its surrounding in the overexpression group. RT-qPCR results showed that the relative transcription level of ROP16 mRNA in the overexpression group was 8 023.459 ± 39.325 with green fluorescence, which was higher than that in the blank vector group (5.540 ± 0.001) (F = 83 188, P < 0.01); Western-blotting showed that the relative expression level of ROP16 protein in the overexpression group was 16.349 ± 0.746, which was higher than that in the blank vector group (1.291 ± 0.333) (F = 831.7, P < 0.01). RT-qPCR results showed that the relative transcription levels of pro-inflammatory (M1) factors IL-1β, TNF-α, IL-18, IL-6 and IL-12 in the overexpression group were 0.495 ± 0.002, 0.337 ± 0.007, and 0.378 ± 0.014, 0.474 ± 0.035, and 0.730 ± 0.021, respectively, which were lower than those in the blank vector group (0.994 ± 0.043, 1.165 ± 0.034, 0.943 ± 0.005, 1.153 ± 0.028, 0.926 ± 0.031) (F = 261.7, 536.5, 1 682.0, 225.0, 78.5; P < 0.01); the relative transcription levels of anti-inflammatory (M2) factors IL-10 and TGF-β mRNA were 7.013 ± 0.032 and 1.608 ± 0.024, respectively, which were significantly higher than those in the blank vector group (0.790 ± 0.031, 1.091 ± 0.027) (F = 23 835.0, 200.1, P < 0.01). Western-blotting revealed that that the relative expression levels of Arg-1, pTyr705-STAT3, and pTyr641-STAT6 proteins in the overexpression group were 2.337 ± 0.089, 3.471 ± 0.046, 3.905 ± 0.045, respectively, which were higher than those in the blank vector group (0.871 ± 0.014, 1.482 ± 0.071, 1.514 ± 0.050) (F = 640.8, 1 608.0, 3 528.0, P < 0.01). The flow cytometry results showed that the apoptosis rate in the overexpression group was (2.990 ± 0.042)%, which was lower than that in the control group (6.480 ± 0.071)% and the blank vector group (5.655 ± 0.290)%, respectively (F = 219.7, P < 0.01). Western-blotting showed that the relative expression levels of the pro-apoptotic proteins Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 in the cells of the overexpression group were 0.558 ± 0.005, 0.640 ± 0.011, 0.593 ± 0.026 and 0.453 ± 0.011, respectively, which were lower than those in the blank vector group (0.991 ± 0.010, 0.926 ± 0.006, 0.963 ± 0.012, 0.834 ± 0.008) (F = 2 850.0, 1 200.0, 359.3, 2 337.0, P < 0.01). RT-qPCR showed that the relative transcription levels of pro-apoptotic genes Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 mRNA in the overexpression group were 0.588 ± 0.086, 0.563 ± 0.025, 0.403 ± 0.014 and 0.158 ± 0.008, respectively, which were lower than those in the blank vector group (0.924 ± 0.016, 0.937 ± 0.041, 0.807 ± 0.032, 0.779 ± 0.014) (F = 24.7, 78.6, 154.9, 265.5, P < 0.01); while the relative transcription level of restraining-apoptosis gene Bcl-2 was 3.702 ± 0.362, which was higher than that in the blank vector group (1.186 ± 0.006) (F = 104.1, P < 0.01). Conclusion T. gondii ROP16 protein is stably expressed in ROP16-MH-S cells, mainly located in and around the nucleus,activating STAT3 and STAT6, phosphorylate Tyr705-STAT3 and Tyr641-STAT6, regulating ROP16-MH-S cells towards M2 polarization and thereby suppressing cell apoptosis.

Key words： Toxoplasma gondii; Rhoptry protein 16; Mouse alveolar macrophages cells; Apoptosis; Signal transducer and activator of transcription 3/6

Cite this article

LI Jia-ming , WANG Yi-xuan , YANG Ning-ai , MA Hui-hui , LAN Min , LIU Chun-lan , ZHAO Zhi-jun . Effects of ROP16 protein of Toxoplasma gondii on polarization and apoptosis of MH-S cells and their related mechanisms[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022 , 40(5) : 579 -586 . DOI: 10.12140/j.issn.1000-7423.2022.05.003

References

[1] Kim K,, Weiss LM. Toxoplasma gondii: the model apicomplexan[J]. Int J Parasitol, 2004, 34(3): 423-432.
[2] Zhang Y,, Lai BS,, Juhas M, et al. Toxoplasma gondii secretory proteins and their role in invasion and pathogenesis[J]. Microbiol Res, 2019, 227: 126293.
[3] Zheng B,, Lu SH. Research progress on immune evasion related molecules of Toxoplasma gondii[J]. Chin J Parasitol Parasit Dis, 2012, 30(5): 396-400. (in Chinese)
[3] ( 郑斌,, 陆绍红. 刚地弓形虫免疫逃避相关分子的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 396-400.)
[4] Lima TS,, Lodoen MB. Mechanisms of human innate immune evasion by Toxoplasma gondii[J]. Front Cell Infect Microbiol, 2019, 9: 103.
[5] Liu GZ,, Wang B,, Wang HF. Advances in research of Toxoplasma gondii rhoptry protein ROP16[J]. Chin J Schisto Control, 2015, 27(2): 217-220. (in Chinese)
[5] ( 刘功振,, 王彬,, 王洪法. 弓形虫棒状体蛋白ROP16的研究进展[J]. 中国血吸虫病防治杂志, 2015, 27(2): 217-220.)
[6] Jensen KDC,, Wang Y,, Wojno EDT, et al. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation[J]. Cell Host Microbe, 2011, 9(6): 472-483.
[7] Xu YW,, Xing RX,, Zhang WH, et al. Toxoplasma ROP16 Ⅰ/Ⅲ ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages[J]. World J Gastroenterol, 2019, 25(45): 6634-6652.
[8] Chtanova T,, Schaeffer M,, Han SJ, et al. Dynamics of neutrophil migration in lymph nodes during infection[J]. Immunity, 2008, 29(3): 487-496.
[9] Boothroyd JC. Have it your way: how polymorphic, injected kinases and pseudokinases enable Toxoplasma to subvert host defenses[J]. PLoS Pathog, 2013, 9(4): e1003296.
[10] Su YJ,, Dong H,, Qiao X, et al. Difference of expression profiling of A549 cells induced by Toxoplasma effector ROP16Ⅱ[J]. Chin J Zoonoses, 2018, 34(4): 323-329. (in Chinese)
[10] ( 苏雅静,, 董辉,, 乔霞, 等. 弓形虫ROP16 Ⅱ效应分子对宿主A549细胞基因表达谱的影响[J]. 中国人兽共患病学报, 2018, 34(4): 323-329.)
[11] Ong YC,, Reese ML,, Boothroyd JC. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6[J]. J Biol Chem, 2010, 285(37): 28731-28740.
[12] Butcher BA,, Fox BA,, Rommereim LM, et al. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control[J]. PLoS Pathog, 2011, 7(9): e1002236.
[13] Saeij JPJ,, Coller S,, Boyle JP, et al. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue[J]. Nature, 2007, 445(7125): 324-327.
[14] Yamamoto M,, Standley DM,, Takashima S, et al. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3[J]. J Exp Med, 2009, 206(12): 2747-2760.
[15] Rosowski EE,, Lu D,, Julien L, et al. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein[J]. J Exp Med, 2011, 208(1): 195-212.
[16] Martinez FO,, Gordon S,, Locati M, et al. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression[J]. J Immunol, 2006, 177(10): 7303-7311.
[17] Rutschman R,, Lang R,, Hesse M, et al. Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production[J]. J Immuno, 2001, 166(4): 2173-2177.
[18] Kochanowsky JA,, Thomas KK,, Koshy AA. ROP16-mediated activation of STAT6 suppresses host cell reactive oxygen species production, facilitating type Ⅲ Toxoplasma gondii growth and survival[J]. mBio, 2021, 12(2): e03305-e03320.
[19] Rutschman R,, Lang R,, Hesse M, et al. Cutting edge: STAT6-dependent substrate depletion regulates nitric oxide production[J]. J Immunol, 2001, 166(4): 2173-2177.
[20] Robben P M,, Mordue D G,, Truscott S M, et al. Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype[J]. J Immunol, 2004, 172(6): 3686-3694.
[21] Modolell M,, Corraliza IM,, Link F, et al. Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by Th1 and Th2 cytokines[J]. Eur J Immunol, 1995, 25(4): 1101-1104.
[22] Alvarez C,, De-La-Torre A,, Vargas M, et al. Striking divergence in Toxoplasma ROP16 nucleotide sequences from human and meat samples[J]. J Infect Dis, 2014, 211(12): 2006-2013.
[23] Han M,, Wu H. Research progress of toxoplasmosis in China[J]. Med Inf, 2018, 31(2): 33-36. (in Chinese)
[23] ( 韩梅,, 吴寒. 我国弓形虫病研究进展[J]. 医学信息, 2018, 31(2): 33-36.)
[24] Zhu Y,, Yang QL. Interaction between Toxoplasma gondii and host cell[J]. Prog Microbiol Immunol, 2014, 42(5): 77-80. (in Chinese)
[24] ( 朱勇,, 杨秋林. 弓形虫与宿主细胞相互作用研究进展[J]. 微生物学免疫学进展, 2014, 42(5): 77-80.)
[25] Carmen JC,, Hardi L,, Sinai AP. Toxoplasma gondii inhibits ultraviolet light-induced apoptosis through multiple interactions with the mitochondrion-dependent programmed cell death pathway[J]. Cell Microbiol, 2006, 8(2): 301-315.
[26] Jia ZH,, Jia Y,, Guo FJ, et al. Phosphorylation of STAT3 at Tyr705 regulates MMP-9 production in epithelial ovarian cancer[J]. PLoS One, 2017, 12(8): e0183622.
[27] Hirano T,, Ishihara K,, Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors[J]. Oncogene, 2000, 19(21): 2548-2556.
[28] Nash PB,, Purner MB,, Leon RP, et al. Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis[J]. J Immunol, 1998, 160(4): 1824-1830.
[29] Earnshaw WC,, Martins LM,, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis[J]. Annu Rev Biochem, 1999, 68(1): 383-424.
Options
Outlines

/

〈 〉