The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen

doi:10.12140/j.issn.1000-7423.2020.01.005

Abstract

Abstract: Objective To investigate the effect of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in livers and spleens of C57BL/6 mice. Methods Thirty-two C57BL/6 mice were randomly assigned into four groups of eight animals each: control group, low-dosage group (50 protoscoleces/mouse), mid-dosage group (500 protoscoleces/mouse) and high-dosage group (2 000 protoscoleces/mouse). The mice received portal vein injection of 200 μl protoscolex suspension or normal saline. Four weeks after infection, four mice in each group were euthanized to collect liver tissues for histopathological observation with routine Masson staining, while the other four mice were euthanized for orbital blood collection and liver and spleen cell suspension preparation. The numbers of CD3⁺ T cells, B cells, NK cells, NKT cells, CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD69⁺ T cells, and CD8⁺CD69⁺ T cells, as well as the percentages of naive CD4⁺ T cells (CD4⁺ Tn), effector memory CD4⁺ T cells (CD4⁺ Tem), naive CD8⁺ T cells (CD8⁺ Tn), effector memory CD8⁺ T cells (CD8⁺ Tem) and central memory CD8⁺ T cells (CD8⁺ Tcm) were detected by flow cytometry (FCM). The FCM data were analyzed with Flow-Jo software. Statistical analysis was performed using Graphad Prism 7.0 software. Results Four weeks after infection, there were no significant changes in the livers of the control mice. Both the low- and mid-dosage groups showed weakening of acute inflammatory cell infiltration in liver tissues, and some lesions had gradually transformed to the fibrotic repair stage, with formation of small granulomatous nodules and appearance of penctate or focal necrosis. In the high-dosage group, small vesicles were seen in the liver. Germinal layers were observed, accompanied by intensive inflammatory cell infiltration. The numbers of CD3⁺ T cells in mouse liver in the high-, mid- and low-dosage groups and the control group were (4.10 ± 0.04) × 10⁵, (3.81 ± 0.14) × 10⁵, (3.02 ± 0.12) × 10⁵, and (2.98 ± 0.03) × 10⁵, respectively, with that in the high-dosage group being significantly higher than the other 3 groups (P < 0.05 or 0.01). The numbers of CD3⁺ T cells in mouse spleen in the high-, mid- and low-dosage groups and the control group were (16.01 ± 0.40) × 10⁵, (11.03 ± 1.21) × 10⁵, (10.10 ± 1.01) × 10⁵, and (9.71 ± 0.90) × 10⁵, respectively (P < 0.01), with that in the high-dosage group being significantly higher than the other 3 groups (P < 0.01). The high-dosage group also had significantly higher numbers of CD4⁺ T cells and CD8⁺ T cells in livers as well as B cells and CD8⁺ T cells in spleens, compared with other groups (P < 0.05 or P < 0.01). The numbers of CD4⁺CD69⁺ T cells in mouse liver in the high-, mid- and low-dosage groups and the control group were (3.23 ± 0.10) × 10⁴, (1.98 ± 0.11) × 10⁴, (1.51 ± 0.26) × 10⁴, and (1.19 ± 0.21) × 10⁴, respectively, with that in the high-dosage group being significantly higher than the other groups (P < 0.05). The respective numbers of CD4⁺CD69⁺ T cells in mouse spleen in the high-, mid- and low-dosage groups and the control group were (10.10 ± 0.41) × 10⁴, (8.91 ± 0.80) × 10⁴, (8.20 ± 0.41) × 10⁴, and (6.81 ± 0.50) × 10⁴, with that in the high-dosage group being significantly higher than the other groups (P < 0.01). The high-dosage group also had significantly higher numbers of CD8⁺CD69⁺ T cells in livers and spleens, compared with other groups (P < 0.05 or P < 0.01). The percentages of CD4⁺ Tn in mouse liver in the high-, mid- and low-dosage groups and the control group were (15.52 ± 1.51)%, (19.3 ± 2.09)%, (20.66 ± 1.28)%, and (23.62 ± 2.84)% respectively, with that in the control group being significantly higher than the other groups (P < 0.05 or P < 0.01). The control group also had significantly higher percentages of CD8⁺ Tn cells in livers and spleens, compared with other groups (P < 0.05 or P < 0.01). The high-dosage group had significantly higher percentages of CD8⁺ Tcm cells in livers and spleens, compared with other groups (P < 0.05). Conclusion In C57BL/6 mice infected with high-dose E. multilocular protoscoleces, the liver T cell recruitment occurred mainly of CD4⁺ and CD8⁺ T cells, while B cell and CD8⁺ T cell were mainly recruited in spleen.

Key words: Echinococcosis multilocularis, T cells, Early stage, Cytometry

CLC Number:

R532.21

ZHANG Ning, ZHANG Chuan-shan, LI Zhi-de, LI Liang, WANG Hui, TAN Ya-chao, SHANG Lin-lin, WEN Hao. The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(1): 30-35.

References 20

[1]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2).
[2]	Han J, Bao GS, Zhang DQ, et al. A newly discovered epidemic area of Echinococcus multilocularis in west Gansu Province in China[J]. PLoS One, 2015, 10(7): e0132731.
[3]	Aji T, Dong JH, Shao YM, et al. Ex vivo liver resection and autotransplantation as alternative to allotransplantation for end-stage hepatic alveolar echinococcosis[J]. J Hepatol, 2018, 69(5): 1037-1046.
[4]	Liu HD, Wang HB, Fan HN, et al. Alveolar echinococcosis and immune evasion[J]. Chin J Parasitol Parasit Dis, 2018, 36(6): 655-660. (in Chinese)(刘寒冬, 王宏宾, 樊海宁, 等. 多房棘球蚴病的免疫逃避机制[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 655-660.)
[5]	Zhang CS, Shao YM, Yang ST, et al. T-cell tolerance and exhaustion in the clearance of Echinococcus multilocularis: role of inoculum size in a quantitative hepatic experimental model[J]. Sci Rep, 2019, 9: 3424.
[6]	Wang XL, Hu Y, Xu YX, et al. Dynamic changes of lymphocyte differentiation and programmed cell death protein ligand 1 expression in mice infected with Schistosoma japonicum[J]. Chin J Parasitol Parasit Dis, 2018, 36(6): 579-585. (in Chinese)(王晓玲, 胡媛, 徐馀信, 等. 日本血吸虫感染小鼠肝脏和脾脏淋巴细胞及其表面程序性死亡配体1表达动态变化的研究[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 579-585.)
[7]	Vuitton DA, Gottstein B.Echinococcus multilocularis and its intermediate host: a model of parasite-host interplay[J]. J Biomed Biotechnol, 2010, 2010: 1-4.
[8]	Wang J, Gottstein B.Immunoregulation in larval Echinococcus multilocularis infection[J]. Parasite Immunol, 2016, 38(3): 182-192.
[9]	Mejri N, Gottstein B.Echinococcus multilocularis metacestode metabolites contain a cysteine protease that digests eotaxin, a CC pro-inflammatory chemokine[J]. Parasitol Res, 2009, 105(5): 1253-1260.
[10]	La X, Zhang F, Li Y, et al. Upregulation of PD-1 on CD4+CD25+ T cells is associated with immunosuppression in liver of mice infected with Echinococcus multilocularis[J]. Int Immunopharmacol, 2015, 26(2): 357-366.
[11]	Wei XL, Xu Q, Rexiti FL, et al. Dynamic changes of DC and T cell subsets in mice during Echinococcus multilocularis infection[J]. Cent Eur J Immunol, 2014, 39(1): 19-24.
[12]	Wu B, Lv FL.Progress of CD8+ T cell-mediated immune response to Toxoplasma gondii infection[J]. Chin J Parasitol Parasit Dis, 2014, 32(2): 143-147. (in Chinese)(吴斌, 吕芳丽. CD8+T细胞免疫应答在刚地弓形虫感染免疫中的功能研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(2): 143-147.)
[13]	Sun Y, Zheng KY, He X, et al. The phenotype changes of kupffer cells in the progression of hepatic schistosomiasis japonica[J]. Chin J Parasitol Parasit Dis, 2017, 35(3): 224-229. (in Chinese)(孙悦, 郑葵阳, 何兴, 等. 库普弗细胞在小鼠日本血吸虫肝病发展过程中的表型变化[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 224-229.)
[14]	Zhang W, Li J, McManus DP. Concepts in immunology and diagnosis of hydatid disease[J]. Clin Microbiol Rev, 2003, 16(1): 18-36.
[15]	Zhang C, Lin R, Li Z, et al. Immune exhaustion of T cells in alveolar echinococcosis patients and its reversal by blocking checkpoint receptor TIGIT in a murine model[J]. Hepatology, 2019, DOI:10.1002/hep.30896.
[16]	Abulizi A, Shao Y, Aji T, et al. Echinococcus multilocularis inoculation induces NK cell functional decrease through high expression of NKG2A in C57BL/6 mice[J]. BMC Infect Dis, 2019, 19(1): 792.
[17]	Lan B, Zhang J, Lu D, et al. Generation of cancer-specific CD8+CD69+ cells inhibits colon cancer growth[J]. Immunobiology, 2016, 221(1): 1-5.
[18]	Kim MV, Ouyang W, Liao W, et al. The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection[J]. Immunity, 2013, 39(2): 286-297.
[19]	Samji T, Khanna KM.Understanding memory CD8+ T cells[J]. Immunol Lett, 2017, 185: 32-39.
[20]	Zheng L, Hu Y, Wang YJ, et al. Effects of common stimulants on intracellular cytokines and CD62L of splenic CD8+ T cells from mice infected with Schistosoma japonicum[J]. Chin J Parasitol Parasit Dis, 2017, 35(3): 218-223. (in Chinese)(郑力, 胡媛, 王燕娟, 等. 四种刺激剂对日本血吸虫感染小鼠脾脏CD8+ T细胞内细胞因子及表面分子CD62L的影响[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 218-223.)