收稿日期: 2021-09-28
修回日期: 2021-12-30
网络出版日期: 2022-04-20
基金资助
国家自然科学基金(82072307);国家自然科学基金(81772224);国家科技重大专项(2018ZX10713001-004)
Study on the polarization of MDSC stimulated by Echinococcus granulosus protoscolex-derived exosomes in vitro
Received date: 2021-09-28
Revised date: 2021-12-30
Online published: 2022-04-20
Supported by
National Natural Science Foundation of China(82072307);National Natural Science Foundation of China(81772224);National Major Science and Technology Projects of China(2018ZX10713001-004)
目的 探究细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞（MDSC）向M2型巨噬细胞极化的动态变化,并探讨二者协同发挥免疫抑制作用的生物学意义。 方法 体外培养细粒棘球蚴原头节并收集培养上清,超速离心上清获得外泌体。透射电镜观察外泌体形态,蛋白质免疫印迹（Western blotting）检测外泌体蛋白CD9和烯醇化酶的表达。取3只健康BALB/c小鼠,制备股骨髓系细胞,刺激分化为MDSC后,分为实验组、阳性对照组和阴性对照组。于6孔板中每孔加入1×106个MDSC细胞,实验组、阳性对照组和阴性对照组分别加入20 μl外泌体（5 μg）、白细胞介素-4（IL-4）（40 ng）和RPMI 1640培养基进行刺激。于刺激24、48和72 h后,分别收集3组MDSC,采用流式细胞术分析单核型MDSC（M-MDSC）的细胞比例变化及其M2型巨噬细胞分子标志F4/80和CD206的表达情况。采用GraphPad Prism 8.0.2统计学软件进行统计学分析。 结果 透射电镜下可见细粒棘球蚴原头节源外泌体为圆形的膜状结构,直径集中在60~90 nm。Western blotting分析结果显示,外泌体有特异性蛋白CD9和烯醇化酶的表达。外泌体刺激MDSC 24 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为（77.14 ± 0.78）%、（75.74 ± 0.50）%,与阴性对照组的（78.13 ± 0.81）%比较差异均无统计学意义（t = 1.24、3.45,P > 0.05）;刺激48 h后,阴性对照组、实验组和阳性对照组M-MDSC占MDSC的比例分别为（80.47 ± 0.85）%、（78.27 ± 0.69）%和（68.01 ± 1.33）%,其中实验组与阴性对照组比较差异无统计学意义（t = 2.48,P > 0.05）,阳性对照组与阴性对照组比较差异有统计学差异意义（t = 11.20,P < 0.01）;刺激72 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为（50.03 ± 0.59）%和（46.14 ± 0.87）%,均低于阴性对照组的（74.94 ± 1.53）%,差异有统计学意义（t = 21.52、23.18,P < 0.01）。外泌体刺激MDSC 24 h、48 h和72 h后,实验组和阳性对照组M-MDSC上表达M2型巨噬细胞分子标志的比例分别为（11.83 ± 0.06）%、（12.48 ± 0.12）%,（15.11 ± 0.21）%、（15.42 ± 0.27）%和（29.12 ± 1.34）%、（31.83 ± 1.27）%,均高于阴性对照组的（8.52 ± 1.11）%、（8.79 ± 0.35）%和（15.49 ± 0.26）%,差异均有统计学意义（P < 0.05或P < 0.01）。 结论 细粒棘球蚴原头节源外泌体体外可调控M-MDSC向M2型巨噬细胞的极化,且极化随刺激时间进程不断推进。
孙叶挺 , 江楠 , 姜岩岩 , 李腾 , 蒋小凤 , 曹建平 , 沈玉娟 . 细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(2) : 175 -180 . DOI: 10.12140/j.issn.1000-7423.2022.02.007
Objective To investigate the dynamic changes of the polarization of myeloid-derived suppressor cells (MDSC) to M2 macrophages stimulated by Echinococcus granulosus protoscolex-derived exosomes, and to explore the biological significance of their synergistic immunosuppressive effect. Methods The exosomes were collected by ultracentrifugation of supernatant of E. granulosus protoscoleces in vitro culture. The morphology of exosomes was observed by transmission electron microscope, and the expression of exosomal protein CD9 and enolase were detected by Western blotting. Subsequently, MDSC stimulated from myeloid cells of 3 healthy BALB/c mice femoral bone marrow were added at 1 × 106 MDSC per well of a 6-well plate, and divided into experimental group, positive control group and negative control group. 20 μl exosomes (5 μg), 20 μl IL-4 (40 ng) and 20 μl RPMI 1640 medium were added respectively for co-culture. The proportions of monocytic DMSC (M-MDSC) and the expression of M2 macrophages molecular markers F480 and CD206 in co-cultured cells at 24 h, 48 h, and 72 h, were analyzed by Flow cytometry, respectively. Statistical analysis was performed by statistical software GraphPad Prism 8.0.2. Results E. granulosus protoscolex-derived exosomes are of circular membrane structure with a diameter of 60-90 nm by transmission electron microscopy. The expression of specific protein CD9 and enolase in exosomes was detected by Western blotting. The proportions of M-MDSC in MDSC in the negative control, experimental and positive control group were (78.13 ± 0.81)%, (77.14 ± 0.78)%, and (75.74 ± 0.50)%, respectively after 24 h of stimulation by exosomes. The differences were not significant (P > 0.05). The proportions of M-MDSC in the negative control, experimental and positive control group were (80.47 ± 0.85)%, (78.27 ± 0.69)%, and (68.01 ± 1.33)%, respectively, after 48 h of stimulation, showed no significant difference between experimental group and negative control group (P > 0.05), however significant difference was observed between the positive control and the negative control group (P < 0.01). After 72 h, the proportions of M-MDSC in the experimental and positive control groups in MDSC were (50.03 ± 0.59)% and (46.14 ± 0.87)%, which are lower than negative control group (74.94 ± 1.53)%, and the differences were statistically significant (P < 0.001). After stimulation with exosomes for 24 h, 48 h, and 72 h, the proportions of M-MDSC expressing M2 macrophages molecular markers in experimental and positive control group were (11.83 ± 0.06)%, (12.48 ± 0.12)%; (15.11 ± 0.21)%, (15.42 ± 0.27)%; and (29.12 ± 1.34)%, (31.83 ± 1.27)%, respectively, which were higher than those in the negative control group(8.52 ± 1.11)%, (8.79 ± 0.35)%, and (15.49 ± 0.26)%, and all differences were statistically significant (P < 0.05 or P < 0.01). Conclusion E. granulosus protoscolex-derived exosomes could regulate the polarization of M-MDSC to M2 macrophages, and the polarization continued to advance with the stimulation time.
| [1] | Alvarez Rojas CA,, Romig T,, Lightowlers MW. Echinococcus granulosus sensu lato genotypes infecting humans: review of current knowledge[J]. Int J Parasitol, 2014, 44(1): 9-18. |
| [2] | McManus DP. Current status of the genetics and molecular taxonomy of Echinococcus species[J]. Parasitology, 2013, 140(13): 1617-1623. |
| [3] | Grubor NM,, Jovanova-Nesic KD,, Shoenfeld Y. Liver cystic echinococcosis and human host immune and autoimmune follow-up: a review[J]. World J Hepatol, 2017, 9(30): 1176-1189. |
| [4] | Zhou XJ,, Wang W,, Cui F, et al. Myeloid-derived suppressor cells exert immunosuppressive function on the T helper 2 in mice infected with Echinococcus granulosus[J]. Exp Parasitol, 2020, 215: 107917. |
| [5] | Lindau D,, Gielen P,, Kroesen M, et al. The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells[J]. Immunology, 2013, 138(2): 105-115. |
| [6] | Vetsika EK,, Koukos A,, Kotsakis A. Myeloid-derived suppressor cells: major figures that shape the immunosuppressive and angiogenic network in cancer[J]. Cells, 2019, 8(12): 1647. |
| [7] | Parker KH,, Beury DW,, Ostrand-Rosenberg S. Myeloid-derived suppressor cells: critical cells driving immune suppression in the tumor microenvironment[J]. Adv Cancer Res, 2015, 128:95-139. |
| [8] | Mills CD,, Kincaid K,, Alt JM, et al. M-1/M-2 macrophages and the Th1/Th2 paradigm[J]. J Immunol, 2000, 164(12): 6166-6173. |
| [9] | Mosser DM,, Edwards JP. Exploring the full spectrum of macrophage activation[J]. Nat Rev Immunol, 2008, 8(12):958-969. |
| [10] | Funes SC,, Rios M,, Escobar-Vera J, et al. Implications of macrophage polarization in autoimmunity[J]. Immunology, 2018, 154(2): 186-195. |
| [11] | Gordon SR,, Maute RL,, Dulken BW, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity[J]. Nature, 2017, 545(7655): 495-499. |
| [12] | Biswas S,, Mandal G,, Roy Chowdhury S, et al. Exosomes produced by mesenchymal stem cells drive differentiation of myeloid cells into immunosuppressive M2-polarized macrophages in breast cancer[J]. J Immunol, 2019, 203(12): 3447-3460. |
| [13] | Xu QG,, Wang WH,, Zhu B, et al. Optimal conditions for the expansion of mouse myeloid-derived suppressor cells in vitro[J]. Curr Immunol, 2015, 35(2): 140-144. (in Chinese) |
| [13] | (许秋桂,, 王文红,, 朱波, 等. 小鼠髓样抑制细胞体外扩增体系的建立[J]. 现代免疫学, 2015, 35(2): 140-144.) |
| [14] | Fan WX,, Diao YJ,, Ma YY, et al. Comparison of ultracentrifugation and membrane based-affinity column methods in exosome isolation from supernatants of prostate cancer cells[J]. J Mod Lab Med, 2019, 34(3): 6-9. (in Chinese) |
| [14] | (范维肖,, 刁艳君,, 马越云, 等. 超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞培养上清外泌体的方法学比较[J]. 现代检验医学杂志, 2019, 34(3): 6-9.) |
| [15] | Sun YT,, Cao JP,, Shen YJ. Research progress on immunosuppressive function of myeloid-derived suppressor cells and its role in parasitic infection[J]. Chin J Parasitol Parasit Dis, 2021, 39 (4): 505-513. (in Chinese) |
| [15] | (孙叶挺,, 曹建平,, 沈玉娟. 髓源抑制性细胞免疫抑制功能及其在寄生虫感染领域的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 505-513.) |
| [16] | E WJ,, Lu YL,, Zhang LQ, et al. Characteristics and clinical value of macrophage polarization in tissues and serum of patients with hepatic alveolar echinocococosis[J]. J Pract Med, 2020, 36(9): 1198-1202. (in Chinese) |
| [16] | (鄂维建,, 芦永良,, 张灵强, 等. 肝泡型包虫病肝组织和血清中巨噬细胞极化特点及临床意义[J]. 实用医学杂志, 2020, 36(9): 1198-1202.) |
| [17] | Zhang XF,, Gong WC,, Cao SK, et al. Dynamic changes of myeloid-derived suppressor cells and regulatory T cells in livers of mice infected with Echinococcus granulosus[J]. Chin J Schisto Control, 2019, 31(6): 622-627. (in Chinese) |
| [17] | (张小凡,, 巩文词,, 曹胜魁, 等. 细粒棘球绦虫感染小鼠肝脏髓源抑制性细胞与调节性T细胞比例动态变化[J]. 中国血吸虫病防治杂志, 2019, 31(6): 622-627.) |
| [18] | Pritchard A,, Tousif S,, Wang Y, et al. Lung tumor cell-derived exosomes promote M2 macrophage polarization[J]. Cells, 2020, 9(5): 1303. |
| [19] | Santos GB,, Monteiro KM,, da Silva ED, et al. Excretory/secretory products in the Echinococcus granulosus metacestode: is the intermediate host complacent with infection caused by the larval form of the parasite?[J]. Int J Parasitol, 2016, 46(13/14): 843-856. |
| [20] | Siles-Lucas M,, Sánchez-Ovejero C,, González-Sánchez M, et al. Isolation and characterization of exosomes derived from fertile sheep hydatid cysts[J]. Vet Parasitol, 2017, 236: 22-33. |
| [21] | Van Ginderachter JA, Beschin A,, de Baetselier P, et al. Myeloid-derived suppressor cells in parasitic infections[J]. Eur J Immunol, 2010, 40(11): 2976-2985. |
| [22] | Loke P,, MacDonald AS,, Allen JE. Antigen-presenting cells recruited by Brugia malayi induce Th2 differentiation of naïve CD4+ T cells[J]. Eur J Immunol, 2000, 30(4): 1127-1135. |
| [23] | Kong YY,, Fuchsberger M,, Xiang SD, et al. Myeloid derived suppressor cells and their role in diseases[J]. Curr Med Chem, 2013, 20(11): 1437-1444. |
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