收稿日期: 2022-09-06
修回日期: 2023-12-29
网络出版日期: 2023-06-20
基金资助
国家自然科学基金(81960378);贵州省科技厅基础研究计划（黔科合基础-ZK[2023]一般516）;贵州省大学生创新创业训练项目(S202210661169);遵义市校联合基金（遵市科合HZ字[2021]278号）;遵义医科大学学术新苗培养及创新探索专项项目（黔科合平台人才[2020]-004）;遵义医科大学研究生教育创新计划项目(ZYK70);遵义医科大学大学生创新创业训练计划项目(ZYDC2021143)
Effect of excretory-secretory antigen TPx of Cysticercus cellulosae on activation of dendritic cells in piglets
Received date: 2022-09-06
Revised date: 2023-12-29
Online published: 2023-06-20
Supported by
National Natural Science Foundation of China(81960378);Basic Research Program of the Department of Science and Technology of Guizhou Province (Qiankehe Foundation-ZK [2023] General 516);Undergraduate Innovation and Entrepreneurship Training Program of Guizhou Province(S202210661169);Zunyi City School Joint Fund ([2021]278);Special Project of Cultivating New Academic Seedlings and Innovative Exploration of Zunyi Medical University (Qian Sci Con Platform Talent[2020]-004);Project of Zunyi Medical University Graduate Education Innovation(ZYK70);Undergraduate Innovation and Entrepreneurship Training Program of Zunyi Medical University(ZYDC2021143)
目的 探讨猪囊尾蚴排泄分泌抗原（ESA）硫氧还蛋白过氧化物酶（TPx）对仔猪树突状细胞（DC）活化的影响。方法 体外诱导培养健康仔猪髓源DC，培养7 d后，加入终浓度为100 ng/ml的脂多糖（LPS）刺激，继续孵育2 d，分别收集培养未成熟DC（imDC）和成熟DC（mDC），光学显微镜和扫描电镜下观察其培养1~9 d的形态学变化，流式细胞术检测其表面标志物CD1和主要组织相容性复合体Ⅱ（MHC-Ⅱ）的表达情况。另收集培养后7 d的imDC，设阴性对照组、TPx组、排泄分泌抗原（ESA）组、LPS阳性对照组，分别加入RPMI 1640培养基、TPx（50 μg/ml）、ESA（50 μg/ml）、LPS（100 ng/ml）刺激48 h后，流式细胞术检测DC表面标志物MHC-Ⅱ、CD80、CD86的表达情况；ELISA检测DC细胞培养上清中肿瘤坏死因子-α（TNF-α）、白细胞介素6（IL-6）、IL-10、IL-12的分泌水平。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果 光学显微镜下可见，培养第1 天，imDC呈卵圆形，形态单一；随着培养时间延长，DC体积逐渐增大，出现伪足和刺突等，并从卵圆形变为不规则状。扫描电镜下可见，与imDC相比，mDC形态不规则，大致呈长梭形，从胞体辐射出众多长短不一的突起，呈树枝状分布，为典型的树突状结构。流式细胞术检测结果显示，imDC中表达CD1、MHC-Ⅱ的DC占比分别为（0.113 ± 0.005）%、（0.430 ± 0.016）%，低于mDC的（21.400 ± 0.327）%、（21.333 ± 0.450）%（t = 130.341、92.906，均P < 0.05）。TPx组表达MHC-Ⅱ、CD80、CD86的DC占比分别为（15.300 ± 0.245）%、（22.900 ± 0.374）%、（13.033 ± 0.249）%，均低于LPS阳性对照组的（19.000 ± 0.374）%、（31.600 ± 0.082）%、（21.300 ± 0.245）%（t = 11.53、46.32、43.84，均P < 0.05）和ESA组的（18.365 ± 0.618）%、（40.400 ± 0.356）%、（30.300 ± 0.283）%]（t = 9.55、93.17、91.57，均P < 0.05）；TPx组表达MHC-Ⅱ的DC占比高于阴性对照组的（12.133 ± 0.492）%（t = 9.87，P < 0.05）。ELISA检测结果显示，培养上清中，TPx组DC分泌IL-6水平为15.682 ± 0.660，低于阴性对照组的21.041 ± 0.901（t = 6.51，P < 0.05）；分泌TNF-α、IL-6、IL-10和IL-12水平均低于LPS阳性对照组的169.037 ± 7.823、42.118 ± 1.932、34.730 ± 1.772、52.504 ± 2.431（t = 36.79、32.09、13.09、35.05，均P < 0.05）；分泌IL-12水平低于ESA组的23.854 ± 1.020（t = 6.93，P < 0.05）。结论 TPx通过诱导DC表面MHC-Ⅱ高表达、CD80和CD86低表达，以及降低IL-6的分泌水平来介导免疫耐受。
关键词： 猪囊尾蚴; 排泄分泌抗原; 硫氧还蛋白过氧化物酶; 树突状细胞
叶井明 , 何威 , 刘慧媛 , 鱼潇 , 罗波 , 刘美辰 , 周必英 . 猪囊尾蚴排泄分泌抗原TPx对仔猪树突状细胞活化的影响[J]. 中国寄生虫学与寄生虫病杂志, 2023 , 41(3) : 286 -293 . DOI: 10.12140/j.issn.1000-7423.2023.03.004
Objective This study investigates the effect of thioredoxin peroxidase (TPx) in the excretory-secretory antigen (ESA) of Cysticercus cellulosae on activation of dendritic cell (DC) in piglets. Methods Healthy piglet medulla-derived DC were cultured in vitroo, in which lipopolysaccharide (LPS) was added at a final concentration of 100 ng/ml on 7 d for stimulation for 2 days, and then continuously cultured for 2 more days to collect immature DC (imDC) and mature DC (mDC) separately. The morphological changes of DCs were observed by light microscopy and scanning electron microscopy on 1 to 9 d of culture. The expression of surface markers CD1 and major histocompatibility complex (MHC-Ⅱ) was detected by flow cytometry. The 7 d imDC was used in the assay with the assigned groups of negative control, TPx, ESA and LPS positive control, to which RPMI 1640 medium, TPx (50 μg/ml), ESA (50 μg/ml) and LPS (100 ng/ml) was added, respectively, to stimulate for 48 h for examining the expression of DC surface markers MHC-Ⅱ, CD80 and CD86 using flow cytometry and for detecting secretion levels of tumor necrosis factor-α (TNF-α), interleukin (IL-6), IL-10, IL-12 in DC culture supernatant by ELISA. One-way ANOVA was used for comparison between multiple groups, and LSD-t test was used for two-way comparison between groups. Results Under ligth microscope, imDC were ovoid in shape with single form at the first day of culture; with the extension of culture time, DC increased in size, appeared pseudopods and spines and other features, and changed from ovoid to irregular shape. Scanning electron microscopy showed that compared with imDC, mDC had irregular morphology, roughly long shuttle shape, and numerous protrusions of different lengths radiating from the cytosol, which were distributed in a dendritic pattern, a typical dendritic structure. Flow cytometry showed that the expression of CD1 and MHC-Ⅱ in imDC was (0.113 ± 0.005)% and (0.430 ± 0.016)%, respectively, which was lower than that of mDC (21.400 ± 0.327)% and (21.333 ± 0.450)% (t = 130.341, 92.906, both P < 0.05). The expression levels of MHC-Ⅱ, CD80, and CD86 in the TPx group were (15.300 ± 0.245)%, (22.900 ± 0.374)% and (13.033 ± 0.249)%, respectively, which were lower than those in the LPS positive control group (19.000 ± 0.374)%, (31.600 ± 0.082)%, and (21.300 ± 0.245)% (t = 11.53, 46.32, 43.84, all P < 0.05) and the ESA group (18.365 ± 0.618)%, (40.400 ± 0.356)% and (30.300 ± 0.283)%] (t = 9.55, 93.17, 91.57, all P < 0.05). The MHC-Ⅱ expression level in the TPx group was higher than that of the negative control group (12.133 ± 0.492)% (t = 9.87, P < 0.05). ELISA results showed that IL-6 level in DC of the TPx group was 15.682 ± 0.660, which was ower than that of 21.041 ± 0.901 in the control group (t = 6.51, P < 0.05); TNF-α (35.711 ± 4.196), IL-6, IL-10 (22.216 ± 1.357) and IL-12 (16.799 ± 0.523) were all lower than those of the LPS positive control group 169.037 ± 7.823, 42.118 ± 1.932, 34.730 ± 1.772, 52.504 ± 2.431 (t = 36.79, 32.09, 13.09, 35.05, all P < 0.05); IL-12 level were lower than that of the ESA group at 23.854 ± 1.020 (t = 6.93, P < 0.05). Conclusion TPx mediates immune tolerance by inducing high expression of DC surface molecules MHC-Ⅱ, low expression of CD80 and CD86, and reducing the secretion levels of IL-6.
| [1] | Gonzales I, Rivera JT, Garcia HH. Pathogenesis of Taenia solium taeniasis and cysticercosis[J]. Parasite Immunol, 2016, 38(3): 136-146. |
| [2] | Winkler AS. Neurocysticercosis in sub-Saharan Africa: a review of prevalence, clinical characteristics, diagnosis, and management[J]. Pathog Glob Health, 2012, 106(5): 261-274. |
| [3] | Luo XN, Zheng YD, Cai XP. Epidemic situation and preventive strategies against taeniasis/cysticercosis[J]. J Pathog Biol, 2007, 2(3): 230-232, 238. (in Chinese) |
| (骆学农, 郑亚东, 才学鹏. 猪带绦虫/囊虫病的流行现状及防制策略[J]. 中国病原生物学杂志, 2007, 2(3): 230-232, 238.) | |
| [4] | CystiTeam Group for Epidemiology and Modelling of Taenia solium Taeniasis/Cysticercosis. The World Health Organization 2030 goals for Taenia solium: insights and perspectives from transmission dynamics modelling[J]. Gates Open Res, 2019, 3: 1546. |
| [5] | Braae UC, Hung NM, Satrija F, et al. Porcine cysticercosis (Taenia solium and Taenia asiatica): mapping occurrence and areas potentially at risk in East and Southeast Asia[J]. Parasit Vectors, 2018, 11(1): 613. |
| [6] | Li HZ, Zang XZ, Qian MB, et al. Current status and research progress of cysticercosis[J]. Chin J Schisto Control, 2018, 30(1): 99-103. (in Chinese) |
| (李焕璋, 臧新中, 钱门宝, 等. 囊尾蚴病流行现况及研究进展[J]. 中国血吸虫病防治杂志, 2018, 30(1): 99-103.) | |
| [7] | Chen T, Zhao GH, Ning CS, et al. Research progress on epidemiology of cysticercosis in China[J]. Shanghai J Animal Husb Vet Med, 2006(5): 18-20. (in Chinese) |
| (陈同, 赵光辉, 宁长申, 等. 我国囊虫病流行病学研究进展[J]. 上海畜牧兽医通讯, 2006(5): 18-20.) | |
| [8] | Rosales-Mendoza S, Monreal-Escalante E, González-Ortega O, et al. Transplastomic plants yield a multicomponent vaccine against cysticercosis[J]. J Biotechnol, 2018, 266: 124-132. |
| [9] | Fan XM, Zhou BY. Research advances on the relationship between cestode excretory/secretory products and host immune response[J]. Chin J Parasitol Parasit Dis, 2020, 38(1): 128-133. (in Chinese) |
| (范贤敏, 周必英. 绦虫排泄分泌物与宿主免疫效应的相关研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 128-133.) | |
| [10] | Fan X, Zhang Y, Ouyang R, et al. Cysticercus cellulosae regulates T-cell responses and interacts with the host immune system by excreting and secreting antigens[J]. Front Cell Infect Microbiol, 2021, 11: 728222. |
| [11] | Zhang L, Fan XM, Ma Q, et al. Effects of an excretory secretory antigen of cysticercus cellulosae on the maturation and activation of DCs in piglets[J]. J Pathog Biol, 2021, 16(11): 1249-1253. (in Chinese) |
| (张雷, 范贤敏, 马琴, 等. 猪囊尾蚴排泄分泌抗原对仔猪DC成熟活化的影响[J]. 中国病原生物学杂志, 2021, 16(11): 1249-1253.) | |
| [12] | He W, Li LZ, Sun XQ, et al. Screening, validation, T-cell antigenic epitopes prediction and eukaryotic expression of Cysticercus cellulosae excretory-secretory antigen thioredoxin peroxidase protein[J]. J Pathog Biol, 2023, 18(2): 174-179, 184. (in Chinese) |
| (何威, 李丽竹, 孙晓晴, 等. 猪囊尾蚴排泄分泌抗原TPx蛋白的筛选验证、 T细胞抗原表位预测及真核表达[J]. 中国病原生物学杂志, 2023, 18(2): 174-179, 184.) | |
| [13] | He W, Luo B, Zhou BY. Research progress of recombinant thioredoxin peroxidase of important human parasites involved in immunoregulation, immunodiagnosis and immunoprophylaxis[J]. Chin J Endem, 2022, 41(10): 856-860. (in Chinese) |
| (何威, 罗波, 周必英. 人体重要寄生虫重组TPx参与免疫调控、免疫诊断及免疫预防的研究进展[J]. 中华地方病学杂志, 2022, 41(10): 856-860.) | |
| [14] | Guo X, Zhang J, Li R, et al. Molecular cloning and functional characterization of a thioredoxin peroxidase gene in Echinococcus multilocularis[J]. Mol Biochem Parasitol, 2021, 245: 111408. |
| [15] | Li YG. Cloning, expression and antigenicity of thioredoxin peroxidase gene from Taenia polycephala[D]. Lanzhou: Gansu Agricultural University, 2009: 47-48. (in Chinese) |
| (李永光. 多头带绦虫硫氧还蛋白过氧化物酶基因的克隆、 表达及抗原性研究[D]. 兰州: 甘肃农业大学, 2009: 47-48.) | |
| [16] | Hilligan KL, Ronchese F. Antigen presentation by dendritic cells and their instruction of CD4+ T helper cell responses[J]. Cell Mol Immunol, 2020, 17(6): 587-599. |
| [17] | Liu XX, Zhu M, Xu Y, et al. Parasitic infection to dendritic cell subsets[J]. Chin J Zoonoses, 2012, 28(10): 1020-1024. (in Chinese) |
| (刘晓霞, 朱明, 徐琦, 等. 寄生虫感染对树突状细胞亚群的影响[J]. 中国人兽共患病学报, 2012, 28(10): 1020-1024.) | |
| [18] | Falcón C, Carranza F, Martínez FF, et al. Excretory-secretory products (ESP) from Fasciola hepatica induce tolerogenic properties in myeloid dendritic cells[J]. Vet Immunol Immunopathol, 2010, 137(1/2): 36-46. |
| [19] | Li LZ. Effect of proteomics-basted cysticercus cellulosae excretory secretory antigen LRRC15 protein on T-cell immune response in piglets[D]. Zunyi: Zunyi Medical University, 2022: 52-55. (in Chinese) |
| (李丽竹. 基于蛋白质组学研究猪囊尾蚴排泄分泌抗原LRRC15蛋白对仔猪T细胞免疫应答的影响[D]. 遵义: 遵义医科大学, 2022: 52-55.) | |
| [20] | White RR, Artavanis-Tsakonas K. How helminths use excretory secretory fractions to modulate dendritic cells[J]. Virulence, 2012, 3(7): 668-677. |
| [21] | Wang Y, Zhou H, Shen Y, et al. Impairment of dendritic cell function and induction of CD4+CD25+Foxp3+ T cells by excretory-secretory products: a potential mechanism of immune evasion adopted by Echinococcus granulosus[J]. BMC Immunol, 2015, 16: 44. |
| [22] | Li Y. Prokaryotic expression of GAPDH gene and Tpx gene of Baylisascaris schroederi and the evaluation of diagnostic value of recombinant antigens[D]. Yaan: Sichuan Agricultural University, 2017: 6-8. (in Chinese) |
| (李宇. 西氏贝蛔虫GAPDH基因和Tpx基因的原核表达与重组抗原诊断价值的评估[D]. 雅安: 四川农业大学, 2017: 6-8.) | |
| [23] | Yin C, Luo XN, Wang S, et al. Prokaryotic expression and biological properties of thioredoxin peroxidase from Taenia solium[J]. Acta Vet Zootechnica Sin, 2014, 45(9): 1512-1517. (in Chinese) |
| (尹才, 骆学农, 王帅, 等. 猪带绦虫硫氧还蛋白过氧化物酶的原核表达及其生物学特性分析[J]. 畜牧兽医学报, 2014, 45(9): 1512-1517.) | |
| [24] | He W, Sun X, Luo B, et al. Regulation of piglet T-cell immune responses by thioredoxin peroxidase from Cysticercus cellulosaeexcretory-secretory antigens[J]. Front Microbiol, 2022, 13: 1019810. |
| [25] | Moll H. Dendritic cells and host resistance to infection[J]. Cell Microbiol, 2003, 5(8): 493-500. |
| [26] | Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells[J]. Annu Rev Immunol, 2003, 21: 685-711. |
| [27] | Sun LY, Ding Z, Chen P, et al. Research progress on tolerogenic dendritic cells[J]. Life Sci Res, 2020, 24(4): 314-320. (in Chinese) |
| (孙庐云, 丁喆, 陈鹏, 等. 耐受性树突状细胞的研究进展[J]. 生命科学研究, 2020, 24(4): 314-320.) | |
| [28] | Maldonado RA, von Andrian UH. How tolerogenic dendritic cells induce regulatory T cells[J]. Adv Immunol, 2010, 108: 111-165. |
| [29] | Xu HY, He XZ. Research progress of tolerant dendritic cells[J]. Organ Transplant, 2014, 5(1): 49-53. (in Chinese) |
| (徐海燕, 何小舟. 耐受性树突状细胞的研究进展[J]. 器官移植, 2014, 5(1): 49-53.) | |
| [30] | Na H, Cho M, Chung Y. Regulation of Th2 cell immunity by dendritic cells[J]. Immune Netw, 2016, 16(1): 1-12. |
| [31] | Dong LY. Various phenotype dendritic cells of rats mediated immune tolerance of liver transplantation in vitro[D]. Kungming: Kunming Medical University, 2012: 31-32. (in Chinese) |
| (董丽英. 大鼠不同表型树突状细胞介导肝移植免疫耐受的体外实验研究[D]. 昆明: 昆明医科大学, 2012: 31-32.) | |
| [32] | Cvetkovic J, Ilic N, Gruden-Movsesijan A, et al. DC-SIGN signalling induced by Trichinella spiralis products contributes to the tolerogenic signatures of human dendritic cells[J]. Sci Rep, 2020, 10(1): 20283. |
| [33] | Lamendour L, Deluce-Kakwata-Nkor N, Mouline C, et al. Tethering innate surface receptors on dendritic cells: a new avenue for immune tolerance induction?[J]. Int J Mol Sci, 2020, 21(15): 5259. |
| [34] | Nam JH, Lee JH, Choi SY, et al. Functional ambivalence of dendritic cells: tolerogenicity and immunogenicity[J]. Int J Mol Sci, 2021, 22(9): 4430. |
| [35] | Qiao YC, Shen J, He L, et al. Changes of regulatory T cells and of proinflammatory and immunosuppressive cytokines in patients with type 2 diabetes mellitus: a systematic review and meta-analysis[J]. J Diabetes Res, 2016, 2016: 3694957. |
| [36] | Qiao YC, Pan YH, Ling W, et al. The Yin and Yang of regulatory T cell and therapy progress in autoimmune disease[J]. Autoimmun Rev, 2017, 16(10): 1058-1070. |
| [37] | Korn T, Hiltensperger M. Role of IL-6 in the commitment of T cell subsets[J]. Cytokine, 2021, 146: 155654. |
| [38] | Yang XO, Nurieva R, Martinez GJ, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs[J]. Immunity, 2008, 29(1): 44-56. |
| [39] | Samanta A, Li B, Song X, et al. TGF-beta and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3[J]. Proc Natl Acad Sci USA, 2008, 105(37): 14023-14027. |
| [40] | Essig K, Hu D, Guimaraes JC, et al. Roquin suppresses the PI3K-mTOR signaling pathway to inhibit T helper cell differentiation and conversion of Treg to Tfr cells[J]. Immunity, 2017, 47(6): 1067-1082. e12. |
| [41] | Svensson MN, Doody KM, Schmiedel BJ, et al. Reduced expression of phosphatase PTPN2 promotes pathogenic conversion of Tregs in autoimmunity[J]. J Clin Invest, 2019, 129(3): 1193-1210. |
| [42] | Li D, Kong C, Tsun A, et al. MiR-125a-5p decreases the sensitivity of Treg cells toward IL-6-mediated conversion by inhibiting IL-6R and STAT3 expression[J]. Sci Rep, 2015, 5: 14615. |
| [43] | Hsieh WC, Hsu TS, Chang YJ, et al. IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells[J]. Nat Commun, 2018, 9(1): 463. |
| [44] | Zhang CK. Study on immune tolerance induced by bone marrow mesenchymal stem cells combined with IL-6 monoclonal antibody in heart transplantation[D]. Suzhou: Soochow University, 2012: 41-43. (in Chinese) |
| (章传凯. 骨髓间充质干细胞联合IL-6单克隆抗体诱导心脏移植免疫耐受的研究[D]. 苏州: 苏州大学, 2012: 41-43.) |
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