Affect of Echinococcus multilocularis protein-mediated NK cell surface receptor NKG2A on the function of NK cells

doi:10.12140/j.issn.1000-7423.2022.01.005

Abstract

Abstract:

Objective To investigate the affect of Echinococcus multilocularis protein mediated natural killer (NK) cell surface inhibitory receptor NK cell lectin-like receptor subfamily C member A (NKG2A) on the function of NK cells. Methods Peripheral blood samples were colleted from the participants for NK cell purification. An aliquate of 0.3 × 10 ⁶ NK cells were resuspended in 100 μl RMPI 1640 medium, which was transferred into a 96-well plate. Four test groups were assigned, including blank control, negative control, E. multilocularia protein (Emp) group, and transforming growth factor-β 1(TGF-β1) group (positive control group). The blank conrol group underwent no further treatment. For the negative control group, 1 μl interleukin-12 (IL-12) and IL-15 (of 1 μg/ml each) were added, while the Emp group was treated with 1 μl IL-12 and IL-15 (at 1 μg/ml each) and 2.5 μl Emp (7 081 μg/ml); to the TGF-β1 group, 1 μl TGF-β1 (1 μg/ml) were added. RPMI 1640 medium was used to adjust the wells to a final volume of 104.5 μl when appropriate. Flow cytometry analysis was used to quantify the expression of NKG2A on NK cells and functional changes of NK cells and NKG2A ⁺NK in secretion of cytokines [interferon-γ (IFN-γ), granzyme B, tumor necrosis factor α (TNF-α) and perforin] after culture stimulated for 24 hours in vitro. The data were analyzed using One-way ANOVA for difference analysis, and LSD or Dunnett test for comparison of the difference between groups. Results The percentage of NKG2A⁺NK cells in Emp group and TGF-β1 group were (3.40 ± 1.53)% and (3.00 ± 1.07)%, respectively, which were significantly higher than that in the negative control group (0.70 ± 0.56)% (P < 0.01). In the Emp group, the percentage of NK cells secreting IFN-γ was (42.38 ± 15.94)%, having was no significant difference compared to the negative control group (61.18 ± 7.18)% (P > 0.05). The percentage of NKG2A⁺NK cells secreting IFN-γ was (25.25 ± 11.57)%, which was lower than that in the negative control group (48.88 ± 12.78)% (P < 0.05); the difference in the percentage of NK and NKG2A⁺NK cells secreting granzyme B, TNF-α, and perforin was insignificant between the Emp griyog and negative control secreting NK cells and NKG2A⁺NK cells (P > 0.05). In the TGF-β1 group, the percentage of NK and NKG2A⁺NK cells secreting IFN-γ was (12.77 ± 2.56)% and (15.17 ± 6.34)%, respectively, which were lower than that in the negative control group (P < 0.01); there was no significant difference in the percentage of NK and NKG2A⁺NK cells secreting granzyme B, TNF-α, and perforin was forund between the Emp and negative control. Of the TGF-β1 group, the percentage of NK cells secreting IFN-γ upon stimulation was lower than that in the Emp group, but the difference was not statistically significant (P > 0.05). Conclusion Emp mediates up-regulation of the expression of NK cell surface receptor NKG2A and inhibits the function of NK cells secretging IFN-γ.

Key words: Echinococcus multilocularis, Protein, Natural Killer cell, NKG2A

CLC Number:

R383.33

ABUDUAINI Abulizi, PAIZULA Shalayiadang, TALAITI Tuergan, ZHANG Rui-qing, WANG Hui, ZHANG Chuan-shan, SHAO Ying-mei, TUERGANAILI Aji. Affect of Echinococcus multilocularis protein-mediated NK cell surface receptor NKG2A on the function of NK cells[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(1): 36-42.

Figures/Tables 4

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References 35

[1]	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.)
[2]	Rodrigues V, Cordeiro-da-Silva A, Laforge M, et al. Impairment of T cell function in parasitic infections[J]. PLoS Negl Trop Dis, 2014, 8(2): e2567. doi: 10.1371/journal.pntd.0002567
[3]	Zhang R, Thabet A, Hiob L, et al. Mutual interactions of the apicomplexan parasites Toxoplasma gondii and Eimeria tenella with cultured poultry macrophages[J]. Parasit Vectors, 2018, 11(1): 453. doi: 10.1186/s13071-018-3040-0
[4]	Schmidt S, Tramsen L, Rais B, et al. Natural killer cells as a therapeutic tool for infectious diseases-current status and future perspectives[J]. Oncotarget, 2018, 9(29): 20891-20907. doi: 10.18632/oncotarget.v9i29
[5]	Jin H, Jia Y, Yao Z, et al. Hepatic stellate cell interferes with NK cell regulation of fibrogenesis via curcumin induced senescence of hepatic stellate cell[J]. Cell Signal, 2017, 33: 79-85. doi: 10.1016/j.cellsig.2017.02.006
[6]	Jeong WI, Park O, Suh YG, et al. Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice[J]. Hepatology, 2011, 53(4): 1342-1351. doi: 10.1002/hep.24190
[7]	Zheng B, Yang Y, Han Q, et al. STAT3 directly regulates NKp46 transcription in NK cells of HBeAg-negative CHB patients[J]. J Leukoc Biol, 2019, 106(4): 987-996. doi: 10.1002/jlb.v106.4
[8]	Radaeva S, Sun R, Jaruga B, et al. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners[J]. Gastroenterology, 2006, 130(2): 435-452. doi: 10.1053/j.gastro.2005.10.055
[9]	Melhem A, Muhanna N, Bishara A, et al. Anti-fibrotic activity of NK cells in experimental liver injury through killing of activated HSC[J]. J Hepatol, 2006, 45(1): 60-71. pmid: 16515819
[10]	Krizhanovsky V, Yon M, Dickins RA, et al. Senescence of activated stellate cells limits liver fibrosis[J]. Cell, 2008, 134(4): 657-667. doi: 10.1016/j.cell.2008.06.049 pmid: 18724938
[11]	Muhanna N, Abu Tair L, Doron S, et al. Amelioration of hepatic fibrosis by NK cell activation[J]. Gut, 2011, 60(1): 90-98. doi: 10.1136/gut.2010.211136 pmid: 20660699
[12]	Gur C, Doron S, Kfir-Erenfeld S, et al. NKp46-mediated killing of human and mouse hepatic stellate cells attenuates liver fibrosis[J]. Gut, 2012, 61(6): 885-893. doi: 10.1136/gutjnl-2011-301400
[13]	Wang J, Zhang C, Wei X, et al. TGF-β and TGF-β/Smad signaling in the interactions between Echinococcus multilocularis and its hosts[J]. PLoS One, 2013, 8(2): e55379. doi: 10.1371/journal.pone.0055379
[14]	Zhang CS, Yang ST, Bi XJ, et al. The expression of TGF-β1 and Gadd45γ in liver tissue of patients with AE and its role in liver injury[J]. J Parasit Biol, 2016, 11(10): 908-912. (in Chinese)
	(张传山, 杨舒婷, 毕晓娟, 等. 泡型包虫病患者肝脏组织TGF-β1和Gadd45γ基因的表达及其在肝损伤中的作用研究[J]. 中国病原生物学杂志, 2016, 11(10): 908-912.)
[15]	Strowig T, Brilot F, Münz C. Noncytotoxic functions of NK cells: direct pathogen restriction and assistance to adaptive immunity[J]. J Immunol, 2008, 180(12): 7785-7791. doi: 10.4049/jimmunol.180.12.7785 pmid: 18523242
[16]	Moretta A, Marcenaro E, Parolini S, et al. NK cells at the interface between innate and adaptive immunity[J]. Cell Death Differ, 2008, 15(2): 226-233. pmid: 17541426
[17]	Dong W, Wu X, Ma S, et al. The mechanism of anti-PD-L1 antibody efficacy against PD-L1-negative tumors identifies NK cells expressing PD-L1 as a cytolytic effector[J]. Cancer Discov, 2019, 9(10): 1422-1437. doi: 10.1158/2159-8290.CD-18-1259
[18]	Kiani Z, Bruneau J, Geraghty DE, et al. HLA-F on autologous HIV-infected cells activates primary NK cells expressing the activating killer immunoglobulin-like receptor KIR3DS1[J]. J Virol, 2019, 93(18): e00933-19.
[19]	Zaghi E, Calvi M, Marcenaro E, et al. Targeting NKG2A to elucidate natural killer cell ontogenesis and to develop novel immune-therapeutic strategies in cancer therapy[J]. J Leukoc Biol, 2019, 105(6): 1243-1251. doi: 10.1002/jlb.2019.105.issue-6
[20]	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. doi: 10.1186/s12879-019-4417-1
[21]	Torgerson PR, Torgerson PR, Keller K, et al. The global burden of alveolar echinococcosis[J]. PLoS Negl Trop Dis, 2010, 4(6): e722. doi: 10.1371/journal.pntd.0000722
[22]	Hübner MP, Manfras BJ, Margos MC, et al. Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients[J]. Clin Exp Immunol, 2006, 145(2): 243-251. doi: 10.1111/j.1365-2249.2006.03142.x pmid: 16879243
[23]	Zhang S, Hüe S, Sène D, et al. Expression of major histocompatibility complex class I chain-related molecule A, NKG2D, and transforming growth factor-beta in the liver of humans with alveolar echinococcosis: new actors in the tolerance to parasites?[J]. J Infect Dis, 2008, 197(9): 1341-1349. doi: 10.1086/589525
[24]	Zhang C, Shao Y, Yang S, 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, 2017, 7(1): 11153. doi: 10.1038/s41598-017-11703-1
[25]	Hou XL, Li LH, Li L, et al. Changes in subsets and functional exhaustion of CD4⁺T cells in spleens of mice infected with Echinococcus multilocularis[J]. Chin J Parasitol Parasit Dis, 2020, 38(5): 611-618, 624. (in Chinese)
	(侯昕伶, 李玲慧, 李亮, 等. 多房棘球蚴感染小鼠脾CD4⁺T细胞亚群及其功能耗竭的变化[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 611-618, 624.)
[26]	Jiao YM, Xia H, Wang XM,et al. Therapeutic effect of NK cells stimulated by Toxoplasma gondii excretory/secretory antigens on B16F10 melanoma in mice[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 444-447, 452. (in Chinese)
	(焦玉萌, 夏惠, 王雪梅, 等. 刚地弓形虫排泄分泌抗原刺激NK细胞过继转输对小鼠B16F10黑色素瘤生长的抑制作用[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 444-447, 452.)
[27]	Yang Y, Han Q, Hou Z, et al. Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction[J]. Cell Mol Immunol, 2017, 14(5): 465-475.
[28]	Zhang QF, Yin WW, Xia Y, et al. Liver-infiltrating CD11b^-CD27^- NK subsets account for NK-cell dysfunction in patients with hepatocellular carcinoma and are associated with tumor progression[J]. Cell Mol Immunol, 2017, 14(10): 819-829. doi: 10.1038/cmi.2016.28
[29]	Cai L, Zhang Z, Zhou L, et al. Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients[J]. Clin Immunol, 2008, 129(3): 428-437. doi: 10.1016/j.clim.2008.08.012
[30]	Tatsumi T, Takehara T. Impact of natural killer cells on chronic hepatitis C and hepatocellular carcinoma[J]. Hepatol Res, 2016, 46(5): 416-422. doi: 10.1111/hepr.12619
[31]	Sun C, Sun HY, Zhang C, et al. NK cell receptor imbalance and NK cell dysfunction in HBV infection and hepatocellular carcinoma[J]. Cell Mol Immunol, 2015, 12(3): 292-302. doi: 10.1038/cmi.2014.91
[32]	Vuitton DA. The ambiguous role of immunity in echinococcosis: protection of the host or of the parasite?[J]. Acta Trop, 2003, 85(2): 119-132. doi: 10.1016/S0001-706X(02)00230-9
[33]	Yan C, Wang L, Li B, et al. The expression dynamics of transforming growth factor-β/Smad signaling in the liver fibrosis experimentally caused by Clonorchis sinensis[J]. Parasit Vectors, 2015, 8: 70. doi: 10.1186/s13071-015-0675-y
[34]	Calabrese F, Valente M, Giacometti C, et al. Parenchymal transforming growth factor beta-1: its type Ⅱ receptor and Smad signaling pathway correlate with inflammation and fibrosis in chronic liver disease of viral etiology[J]. J Gastroenterol Hepatol, 2003, 18(11): 1302-1308. doi: 10.1046/j.1440-1746.2003.03162.x
[35]	Shi J, Zhao J, Zhang X, et al. Activated hepatic stellate cells impair NK cell anti-fibrosis capacity through a TGF-β-dependent emperipolesis in HBV cirrhotic patients[J]. Sci Rep, 2017, 7: 44544. doi: 10.1038/srep44544

抗体 Antibody	荧光标记 Fluorescent label	克隆号 Clone number
CD3	PE-Cy7	UCHT1
CD56	APC-Cy7	HCD56
CD16	FITC	3G8
NKG2A	PE	131411
NKG2D	PerCP-Cy5.5	1D11
IFN-γ	APC	4S.B3
TNF-α	PE/Dazzle^TM 594	MAb11
Perforin	Brilliant Violet 510^TM	dG9
Granzyme B	Pacific blue	GB11