刚地弓形虫ROP16蛋白对MH-S细胞极化和凋亡的影响及其相关机制

doi:10.12140/j.issn.1000-7423.2022.05.003

摘要/Abstract

摘要：

目的探究刚地弓形虫棒状体蛋白16（ROP16）在小鼠肺泡巨噬细胞（MH-S细胞）中的表达及其对细胞极化和凋亡的影响和相关机制。方法利用携带绿色荧光标签的ROP16过表达慢病毒转染MH-S细胞,构建稳定表达ROP16的ROP16-MH-S细胞株（过表达组）,同时设空载体慢病毒转染对照组（空载体组）和无转染对照组（对照组）。转染72 h后免疫荧光法检测ROP16-MH-S中ROP16的表达及其在细胞中的定位。以甘油醛-3-磷酸脱氢酶（GAPDH）基因的转录水平为内参照,RT-qPCR检测ROP16-MH-S细胞中促炎（M1）因子白细胞介素-1β（IL-1β）、肿瘤坏死因子（TNF-α）、IL-18、IL-6、IL-12和抗炎（M2）因子IL-10、转化生长因子-β（TGF-β）,以及抑凋亡基因B细胞淋巴瘤/白血病-2（Bcl-2）和促凋亡基因Bcl-2相关X蛋白（Bax）、半胱氨酸蛋白酶3（Caspase 3）、Caspase 9等mRNA的相对转录水平;Western blotting检测ROP16-MH-S细胞中极化蛋白精氨酸酶1（Arg-1）、信号转导和转录激活因子3（STAT3）、705位点磷酸化（pTyr705）-STAT3、STAT6、pTyr641-STAT6和凋亡蛋白Caspase 9、Caspase 3、Bcl-2、Bax蛋白的相对表达水平;流式细胞术检测ROP16-MH-S细胞的凋亡情况。组间比较采用单因素方差分析。结果转染后72 h,空载体组和过表达组90%以上的细胞可见较强的绿色荧光,过表达组细胞中可见明显红色荧光聚集在ROP16-MH-S细胞核及其周围。RT-qPCR结果显示,过表达组ROP16 mRNA的相对转录水平为8 023.459 ± 39.325,高于空载体组（5.540 ± 0.001）（F = 83 188,P < 0.01）;Western blotting检测结果显示,过表达组ROP16蛋白的相对表达水平为16.349 ± 0.746,高于空载体组（1.291 ± 0.333）（F = 831.7,P < 0.01）。RT-qPCR结果显示,过表达组促炎（M1）因子IL-1β、TNF-α、IL-18、IL-6、IL-12 mRNA的相对转录水平分别为0.495 ± 0.002、0.337 ± 0.007、0.378 ± 0.014、0.474 ± 0.035和0.730 ± 0.021,均低于空载体组（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）;抗炎（M2）因子IL-10、TGF-β mRNA的相对转录水平分别为7.013 ± 0.032和1.608 ± 0.024,均高于空载体组（0.790 ± 0.031、1.091 ± 0.027）（F = 23 835.0、200.1,P < 0.01）。Western blotting检测结果显示,过表达组Arg-1、pTyr705-STAT3、pTyr641-STAT6蛋白相对表达水平分别为2.337 ± 0.089、3.471 ± 0.046、3.905 ± 0.045,均高于空载体组（0.871 ± 0.014、1.482 ± 0.071、1.514 ± 0.050）（F = 640.8、1 608.0、3 528.0,P < 0.01）。流式细胞术检测结果显示,过表达组细胞凋亡率为（2.990 ± 0.042）%,低于对照组（6.480 ± 0.071）%和空载体组（5.655 ± 0.290）%（F = 219.7,P < 0.01）。Western blotting检测结果显示,过表达组促凋亡蛋白Bax、Caspase 3、Caspase 9的相对表达水平分别为0.558 ± 0.005、0.640 ± 0.011、0.593 ± 0.026,Bax/Bcl-2为0.453 ± 0.011,均低于空载体组（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检测结果显示,过表达组促凋亡基因Bax、Caspase 3、Caspase 9 mRNA的相对转录水平分别为0.588 ± 0.086、0.563 ± 0.025和0.403 ± 0.014,Bax/Bcl-2为0.158 ± 0.008,均低于空载体组（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）;过表达组抑凋亡基因Bcl-2 mRNA的相对转录水平为3.702 ± 0.362,高于空载体组（1.186 ± 0.006）（F = 104.1,P < 0.01）。结论 ROP16蛋白在ROP16-MH-S细胞中（主要位于细胞核及其周围）稳定表达,可激活STAT3、STAT6,磷酸化Tyr705-STAT3、Tyr641-STAT6,进而调控细胞向M2极化,抑制细胞凋亡。

关键词: 刚地弓形虫, 棒状体蛋白16, 小鼠肺泡巨噬细胞, 凋亡, 信号转导和转录激活因子3/6

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

中图分类号:

R382.5

李佳铭, 王艺璇, 杨宁爱, 马慧慧, 兰敏, 刘春兰, 赵志军. 刚地弓形虫ROP16蛋白对MH-S细胞极化和凋亡的影响及其相关机制[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 579-586.

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.

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参考文献 29

[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.

基因名称 Gene name	引物序列（5′→3′） Primer sequence（5′→3′）
GAPDH	F： GGTTGTCTCCTGCGACTTCA R： TGGTCCAGGGTTTCTTACTCC
Bax	F： TTGCCCTCTTCTACTTTGCTAG R： CCATGATGGTTCTGATCAGCTC
Bcl-2	F： GATGACTTCTCTCGTCGCTAC R： GAACTCAAAGAAGGCCACAATC
Caspase 3	F： AACAAAATGATTCTGTGAGCCC R： TTGACAAATGCTTTTCCCTGAG
Caspase 9	F： TGTGAATATCTTCAACGGGAGC R： GAGTAGGACACAAGGATGTCAC
IL-1β	F： TGGACCTTCCAGGATGAGGACA R： GTTCATCTCGGAGCCTGTAGTG
IL-6	F： CTTGCAAGACTTCCATCCAC R： AGTGGTAGACAGGTCTGTTGG
IL-10	F： ACCTGGTAGAAGTGATGCCCCAGGCA R： CTATGCAGTTGATGAAGATGTCAAA
IL-12	F： TTGAACTGGCGTTGGAAGCACG R： CCACCTGTGAGTTCTTCAAAGGC
IL-18	F： AGACCTGGAATCAGACAACTTT R： TCAGTCATATCCTCGAACACAG
TNF-α	F： CCTGTAGCCCACGTCGTAC R： GGGAGTAGACAAGGTACAACCC
TGF-β	F： ACGTCACTGGAGTTGTACGG R： GGGGCTGATCCCGTTGATT