收稿日期: 2021-08-10
修回日期: 2021-10-20
网络出版日期: 2022-04-16
基金资助
上海市自然科学基金(19ZR1462600);国家自然科学基金面上项目(81971969);国家自然科学基金面上项目(81772225)
Study on the inhibitory effect of natural killer cells on liver fibrosis of schistosomiasis
Received date: 2021-08-10
Revised date: 2021-10-20
Online published: 2022-04-16
Supported by
Natural Science Foundation of Shanghai(19ZR1462600);Surface Project of National Natural Science Foundation of China(81971969);Surface Project of National Natural Science Foundation of China(81772225)
目的 通过单抗敲低或过继自然杀伤（NK）细胞,探讨NK细胞在血吸虫病肝纤维化中的作用。 方法 24只C57BL/6健康小鼠取肝脏,制备NK细胞,用白细胞介素-15（IL-15）、IL-2、IL-18活化NK细胞。40只C57BL/6健康小鼠随机分为单抗组、IgG组、生理盐水组、NK细胞组、PBS组等5组,每组8只,每鼠经腹部贴片感染日本血吸虫尾蚴（20 ± 1）条。单抗组、IgG组和生理盐水组小鼠于感染前1周开始,每周分别尾静脉注射1次抗NK1.1单抗（100 μg/鼠）、IgG（100 μg/鼠）、生理盐水,每次200 μl/鼠;NK细胞组和PBS组小鼠于感染后4周开始,每周分别尾静脉注射1次活化的NK细胞（1 × 105/鼠）、PBS,每次200 μl/鼠。感染后6和8周,分别取各组小鼠肝脏,流式细胞术检测小鼠肝NK细胞比例变化,Masson染色观察肝组织纤维化变化,荧光定量PCR检测Ⅰ型胶原蛋白（Ⅰ-C）、Ⅲ-C、Ⅳ-C、层黏连蛋白（LN）、纤维连接蛋白（FN）mRNA相对转录水平等肝纤维化指标。 结果 感染后6周,单抗组肝NK细胞比例为（2.56 ± 0.47）%,低于IgG组的（4.75 ± 0.62）%和生理盐水组的（5.35 ± 0.40）%（F = 31.59,P < 0.01）;感染后8周,单抗组、IgG组和生理盐水组NK细胞比例分别为（2.65 ± 0.23）%、（3.43 ± 0.46）%和（3.56 ± 0.46）%,差异无统计学意义（F = 4.48,P > 0.05）。感染后6、8周,NK细胞组肝NK细胞比例分别为（5.58 ± 0.30）%、（3.73 ± 0.42）%,与PBS组的（5.51 ± 0.27）%、（2.32 ± 0.40）%差异均无统计学意义（t = 0.25、2.64,P > 0.05）。感染后6、8周,单抗组肝纤维化面积分别为（8.42 ± 1.30）× 104、（9.55 ± 1.55）× 104 μm2,均大于IgG组的（6.40 ± 1.40）× 104、（6.11 ± 1.10）× 104 μm2和生理盐水组的（6.73 ± 1.61）× 104、（6.62 ± 1.60）× 104 μm2（F = 13.63、30.33,P < 0.01）。感染后6周,NK细胞组肝纤维化面积为（5.97 ± 0.96）× 104 μm2,小于PBS组的（8.27 ± 1.62）× 104 μm2（t = 4.85,P < 0.01）;感染后8周,NK细胞组、PBS组肝纤维化面积分别为（6.33 ± 0.98）× 104 μm2、（6.97 ± 1.11）× 104 μm2,差异无统计学意义（t = 1.80,P > 0.05）。感染后6周,单抗组Ⅳ-C、LN、FN mRNA相对转录水平分别为1.81 ± 0.47、1.63 ± 0.38和1.41 ± 0.16,较IgG组的1.00 ± 0.35、0.81 ± 0.29、0.79 ± 0.16和生理盐水组的1.00 ± 0.12、1.00 ± 0.10、1.00 ± 0.14均上调（F = 8.30、8.25、14.40,P < 0.01）;感染后8周,单抗组Ⅳ-C mRNA相对转录水平为1.66 ± 0.21,较IgG组的0.94 ± 0.26和生理盐水组的1.00 ± 0.09均上调（F = 15.95,P < 0.01）。感染后6周,NK细胞组Ⅰ-C、Ⅲ-C、LN mRNA相对转录水平分别为0.66 ± 0.12、0.61 ± 0.06、0.64 ± 0.09,较PBS组的1.01 ± 0.14、1.01 ± 0.14、1.01 ± 0.16均下调（t = 3.36、4.41、3.59,P < 0.05）;感染后8周,NK细胞组Ⅰ-C、Ⅲ-C、LN mRNA相对转录水平分别为0.82 ± 0.40、0.93 ± 0.37、0.73 ± 0.30,与PBS组的1.04 ± 0.38、1.02 ± 0.25、1.06 ± 0.49相比,差异无统计学意义（t = 0.55、0.27、0.81,P > 0.05）。 结论 小鼠感染日本血吸虫后肝NK细胞在肝纤维化形成早期可发挥抑制肝纤维化的作用。
高元 , 章孝成 , 胡媛 , 曹建平 . 自然杀伤细胞抑制血吸虫病肝纤维化作用的研究[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(2) : 168 -174 . DOI: 10.12140/j.issn.1000-7423.2022.02.006
Objective Using monoclonal antibodies to deplete or passively transfer natural killer (NK) cell, to explore the role of NK cells in schistosomiasis liver fibrosis. Methods The livers of 24 C57BL/6 healthy mice were collected to isolate NK cells, and the NK cells were activated with interleukin-15 (IL-15), IL-2 and IL-18. Forty C57BL/6 mice were randomly divided into five groups, namely monoclonal antibody group, IgG group, normal saline group, NK cell group and PBS group, with 8 mice in each group. Each mouse was infected with Schistosoma japonicum cercariae (20 ± 1) via an abdominal patch. The mice in the monoclonal antibody group, IgG group and normal saline group were injected with anti-NK1.1 monoclonal antibody (100 μg/mouse), IgG (100 μg/mouse), and normal saline through the tail vein at a dose of 200 μl/mouse each time, once a week, started one week before the infection. Four weeks after infection, the mice in the NK cell group and PBS group were transferred with activated NK cells (1 × 105/mouse) and PBS through the tail vein weekly at dose of 200 μl/mouse each time. At six and eight weeks after infection, flow cytometry was used to detect changes in the proportion of NK cells in the liver of the mice. Masson staining was used to observe liver tissue lesions, and Real-time PCR was used to detect indicators of liver fibrosis, including mRNA relative transcription levels of Ⅰ-collagen (Ⅰ-C), Ⅲ-C, Ⅳ-C, fibronectin (FN), and laminin (LN). Results The proportion of liver NK cells at six weeks after infection in the monoclonal antibody group was (2.56 ± 0.47)%, which was lower than that in the IgG group (4.75 ± 0.62)% and normal saline group (5.35 ± 0.40)% (F = 31.59, P < 0.01). At 8 weeks post-infection, the proportion of liver NK cells were (2.65 ± 0.23)%, (3.43 ± 0.46)%, and (3.56 ± 0.46)% for the monoclonal antibody group, the IgG group and the normal saline group, respectively, and the difference was not statistically significant (F = 4.48, P > 0.05). The proportion of liver NK cells in the NK cell group was (5.58 ± 0.30)% and (3.73 ± 0.42)% for 6 and 8 weeks after infection, respectively. There was no statistically significant difference between 6 weeks (5.51 ± 0.27)% and 8 weeks (2.32 ± 0.40)% after infection for the PBS group (t = 0.25, 2.64, P > 0.05). The area of liver fibrosis in the monoclonal antibody group was (8.42 ± 1.30) × 104 μm2 at 6 weeks after infection, which was larger than that in the IgG group (6.40 ± 1.40) × 104 μm2 and normal saline group (6.73 ± 1.61) × 104 μm2 (F = 13.63, P < 0.01). The area of liver fibrosis for the monoclonal antibody group was (9.55 ± 1.55) × 104 μm2 at 8 weeks after infection, which was larger than that in the IgG group (6.11 ± 1.10) × 104 μm2 and normal saline group (6.62 ± 1.60) × 104 μm2 (F = 30.33, P < 0.01). The area of liver fibrosis in the NK cell group was (5.97 ± 0.96) × 104 μm2 at 6 weeks after infection, which was smaller than that in the PBS group (8.27 ± 1.62) × 104 μm2 (t = 4.85, P < 0.01). However, at 8 weeks after infection, the area of liver fibrosis for the NK cell group and the PBS group were (6.33 ± 0.98) × 104 μm2 and (6.97 ± 1.11) × 104 μm2, respectively. The difference was not statistically significant (t = 1.80, P > 0.05). The relative transcription levels of Ⅳ-C, FN, and LN mRNA in the monoclonal antibody group were 1.81 ± 0.47, 1.63 ± 0.38 and 1.41 ± 0.16, which are higher than that in the IgG group (1.00 ± 0.35, 0.81 ± 0.29, 0.79 ± 0.16) and the normal saline group (1.00 ± 0.12, 1.00 ± 0.10, 1.00 ± 0.14) (F = 8.30, 8.25, 14.40, P < 0.01). At 8 weeks after infection, the relative transcription level of Ⅳ-C mRNA in the monoclonal antibody group was 1.66 ± 0.21, which is higher than that in the IgG group (0.94 ± 0.26) and the saline group (1.00 ± 0.09) (F = 15.95, P < 0.01). At 6 weeks after infection, the relative transcription levels for Ⅰ-C, Ⅲ-C and LN mRNA in the NK cell group were 0.66 ± 0.12, 0.61 ± 0.06, 0.64 ± 0.09, respectively. The relative transcription levels were much higher compared with the PBS group (1.01% ± 0.14, 1.01 ± 0.14, 1.01 ± 0.16) (t = 3.36, 4.41, 3.59, P < 0.05). No statistical significant differences were observed at 8 weeks after infection for the NK cell group (0.82 ± 0.40, 0.93 ± 0.37, 0.73 ± 0.30) and the PBS group (1.04 ± 0.38, 1.02 ± 0.25, 1.06 ± 0.49) (t = 0.55, 0.27, 0.81, P > 0.05). Conclusion It was shown that after being infected with S. japonicum, NK cells from mouse liver might inhibit liver fibrosis in the early stage of its formation.
| [1] | McManus DP,, Bergquist R,, Cai PF, et al. Schistosomiasis-from immunopathology to vaccines[J]. Semin Immunopathol, 2020, 42(3): 355-371. |
| [2] | Zhang LJ,, Xu ZM,, Yang F, et al. Endemic status of schistosomiasis in People’s Republic of China in 2020[J]. Chin J Schisto Control, 2021, 33(3): 225-233. (in Chinese) |
| [2] | (张利娟,, 徐志敏,, 杨帆, 等. 2020年全国血吸虫病疫情通报[J]. 中国血吸虫病防治杂志, 2021, 33(3): 225-233.) |
| [3] | Wang L,, Wang YH,, Quan J. Exosomes derived from natural killer cells inhibit hepatic stellate cell activation and liver fibrosis[J]. Hum Cell, 2020, 33(3): 582-589. |
| [4] | van Eeden C,, Khan L,, Osman MS, et al. Natural killer cell dysfunction and its role in COVID-19[J]. Int J Mol Sci, 2020, 21(17): 6351. |
| [5] | Wijaya RS,, Read SA,, Schibeci S, et al. KLRG1+ natural killer cells exert a novel antifibrotic function in chronic hepatitis B[J]. J Hepatol, 2019, 71(2): 252-264. |
| [6] | Yi HS,, Lee YS,, Byun JS, et al. Alcohol dehydrogenase Ⅲ exacerbates liver fibrosis by enhancing stellate cell activation and suppressing natural killer cells in mice[J]. Hepatology, 2014, 60(3): 1044-1053. |
| [7] | Fan YT,, Zhang WD,, Wei HM, et al. Hepatic NK cells attenuate fibrosis progression of non-alcoholic steatohepatitis in dependent of CXCL10-mediated recruitment[J]. Liver Int, 2020, 40(3): 598-608. |
| [8] | Hou X,, Yu FZ,, Man SQ, et al. Negative regulation of Schistosoma japonicum egg-induced liver fibrosis by natural killer cells[J]. PLoS Negl Trop Dis, 2012, 6(1): e1456. |
| [9] | Villesen IF,, Daniels SJ,, Leeming DJ, et al. Review article: the signalling and functional role of the extracellular matrix in the development of liver fibrosis[J]. Aliment Pharmacol Ther, 2020, 52(1): 85-97. |
| [10] | Jiang PY,, Pan WQ. Advances in liver fibrosis and pathogenesis of schistosomiasis[J]. Chin Trop Med, 2018, 18(8): 847-852. (in Chinese) |
| [10] | (姜鹏月,, 潘卫庆. 血吸虫病肝纤维化及其致病机制研究进展[J]. 中国热带医学, 2018, 18(8): 847-852.) |
| [11] | Hu Y,, Wang XL,, Wei YH, et al. Functional inhibition of natural killer cells in a BALB/c mouse model of liver fibrosis induced by Schistosoma japonicum infection[J]. Front Cell Infect Microbiol, 2020, 10: 598987. |
| [12] | Terrén I,, Orrantia A,, Vitallé J, et al. NK cell metabolism and tumor microenvironment[J]. Front Immunol, 2019, 10: 2278. |
| [13] | Becker PSA,, Suck G,, Nowakowska P, et al. Selection and expansion of natural killer cells for NK cell-based immunotherapy[J]. Cancer Immunol Immunother, 2016, 65(4): 477-484. |
| [14] | Peng H,, Tian ZG. NK cells in liver homeostasis and viral hepatitis[J]. Sci China Life Sci, 2018, 61(12): 1477-1485. |
| [15] | Fasbender F,, Widera A,, Hengstler JG, et al. Natural killer cells and liver fibrosis[J]. Front Immunol, 2016, 7: 19. |
| [16] | Hayakawa Y,, Andrews DM,, Smyth MJ. Subset analysis of human and mouse mature NK cells[J]. Methods Mol Biol, 2010, 612: 27-38. |
| [17] | Widowati W,, Jasaputra DK,, Sumitro SB, et al. Effect of interleukins (IL-2, IL-15, IL-18) on receptors activation and cytotoxic activity of natural killer cells in breast cancer cell[J]. Afr Health Sci, 2020, 20(2): 822-832. |
| [18] | Liu C,, Zhang YS,, Chen F, et al. Immunopathology in schistosomiasis is regulated by TLR2, 4- and IFN-γ-activated MSC through modulating Th1/Th2 responses[J]. Stem Cell Res Ther, 2020, 11(1): 217. |
| [19] | Chester C,, Fritsch K,, Kohrt HE. Natural killer cell immunomodulation: targeting activating, inhibitory, and co-stimulatory receptor signaling for cancer immunotherapy[J]. Front Immunol, 2015, 6: 601. |
| [20] | Khomich O,, Ivanov AV,, Bartosch B. Metabolic hallmarks of hepatic stellate cells in liver fibrosis[J]. Cells, 2019, 9(1): 24. |
| [21] | Hamidzadeh K,, Christensen SM,, Dalby E, et al. Macrophages and the recovery from acute and chronic inflammation[J]. Annu Rev Physiol, 2017, 79: 567-592. |
| [22] | Zhang YX,, Xiong DH,, Li YY, et al. Schistosoma japonicum infection in treg-specific USP21 knockout mice[J]. J Immunol Res, 2021, 2021: 6613162. |
| [23] | Hammerich L,, Tacke F. Emerging roles of myeloid derived suppressor cells in hepatic inflammation and fibrosis[J]. World J Gastrointest Pathophysiol, 2015, 6(3): 43-50. |
| [24] | Park HS,, Kim J,, Cho MY, et al. Effectual labeling of natural killer cells with upconverting nanoparticles by electroporation for in vivo tracking and biodistribution assessment[J]. ACS Appl Mater Interfaces, 2020, 12(44): 49362-49370. |
/
| 〈 |
|
〉 |