干扰素-α介导系统性红斑狼疮外周血CD56dimCD57+自然杀伤细胞功能的损伤

doi:10.19723/j.issn.1671-167X.2023.06.004

摘要/Abstract

摘要：

目的: 探究干扰素-α (interferon-α, IFN-α)对系统性红斑狼疮(systemic lupus erythematosus, SLE)患者外周血CD56^dimCD57⁺自然杀伤(natural killer, NK)细胞凋亡和杀伤功能的调控作用及具体机制。方法: 选取大连医科大学附属第二医院就诊的64例初治SLE患者和16例健康人(healthy control, HC)为研究对象，采用实时荧光定量聚合酶链式反应检测NK细胞杀伤功能相关分子的基因表达水平；将CD56^dimCD57⁺NK细胞与K562细胞共培养后，采用膜联蛋白和7氨基放线菌素标记凋亡的K562细胞；用浓度分别为20、40、80 μmol/L的过氧化氢(hydrogen peroxide, H₂O₂) 处理外周血单个核细胞，并以不使用H₂O₂处理作为对照组，采用流式细胞术检测穿孔素(per-forin, PRF)表达水平；采用酶联免疫吸附试验测定血清中IFN-α的浓度；采用流式细胞术检测CD56^dimCD57⁺NK细胞表面IFN-α受体(interferon-α receptor, IFNAR)表达水平，并用平均荧光强度(mean fluorescence intensity, MFI)表示；用浓度为1 000 U/mL的IFN-α处理CD56^dimCD57⁺NK细胞24、48、72 h，并以不使用IFN-α处理作为对照组，采用流式细胞术检测细胞凋亡及线粒体活性氧(mitochondrial reactive oxygen species, mtROS)表达水平，并用MFI表示。结果: 与HC(n=3)相比，SLE患者(n=3)外周血总NK细胞的PRF1基因表达水平降低(1.24±0.41 vs. 0.57±0.12, P=0.05)。与HC(n=5)相比，SLE患者(n=5)外周血CD56^dimCD57⁺NK细胞杀伤K562细胞的能力明显下降(58.61%±10.60% vs. 36.74%±6.27%, P < 0.01)。与对照组(n=5, 97.51%±1.67%)相比，不同浓度的H₂O₂处理可显著下调CD56^dimCD57⁺NK细胞的PRF表达水平且呈剂量依赖性，浓度为20 μmol/L的H₂O₂组PRF为83.23%±8.48% (n=5, P < 0.05)，浓度为40 μmol/L的H₂O₂组PRF为79.53%±8.56% (n=5, P < 0.01)，浓度为80 μmol/L的H₂O₂组PRF为76.67%±7.16% (n=5，P < 0.01)。与HC (n=16)相比，系统性红斑狼疮疾病活动评分(systemic lupus erythematosus disease activity index, SLEDAI) 为中高疾病活动度(SLEDAI≥10)的SLE患者(n=45)血清IFN-α水平显著增高[(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]。同时与HC (n=6)相比，SLE患者(n=6)外周血CD56^dimCD57⁺NK细胞IFNAR1表达增高(MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05)，与HC (n=6)相比，SLE患者(n=7)外周血CD56^dimCD57⁺NK细胞IFNAR2表达增高(MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05)。与对照组(n=6)相比，IFN-α刺激(n=6)可显著促进CD56^dimCD57⁺NK细胞凋亡(20.48%±7.01% vs. 37.82%±5.84%, P < 0.05)。同时，与对照组(n=4, MFI: 1 049±174.5)相比，不同时间下用IFN-α刺激CD56^dimCD57⁺NK细胞可显著促进mtROS的产生，且呈时间依赖性，48 h的MFI为3 437±1 472 (n=4, P < 0.05)，72 h的MFI为6 495±1 089 (n=4, P < 0.000 1)，而刺激24 h时差异无统计学意义。结论: SLE患者血清中高水平的IFN-α可能通过促进mtROS产生诱导细胞凋亡并抑制PRF表达，下调CD56^dimCD57⁺NK杀伤功能。

关键词: 系统性红斑狼疮, 自然杀伤细胞, 干扰素-α, 活性氧, 穿孔素

Abstract:

Objective: To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56^dimCD57⁺ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism. Methods: A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56^dimCD57⁺ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H₂O₂), and treated without H₂O₂ as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56^dimCD57⁺ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56^dimCD57⁺ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI. Results: Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56^dimCD57⁺ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H₂O₂ treatment significantly down-regulated the PRF expression levels of CD56^dimCD57⁺ NK cells in a dose-dependent manner, the 20 μmol/L H₂O₂ PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H₂O₂ PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H₂O₂ PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56^dimCD57⁺ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56^dimCD57⁺ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56^dimCD57⁺ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56^dimCD57⁺ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation. Conclusion: High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56^dimCD57⁺ NK killing function.

Key words: Systemic lupus erythematosus, Natural killer cells, Interferon-α, Reactive oxygen species, Perforin

中图分类号:

R593.2

赵祥格,刘佳庆,黄会娜,陆智敏,白自然,李霞,祁荆荆. 干扰素-α介导系统性红斑狼疮外周血CD56^dimCD57⁺自然杀伤细胞功能的损伤[J]. 北京大学学报（医学版）, 2023, 55(6): 975-981.

Xiang-ge ZHAO,Jia-qing LIU,Hui-na HUANG,Zhi-min LU,Zi-ran BAI,Xia LI,Jing-jing QI. Interferon-α mediating the functional damage of CD56^dimCD57⁺natural killer cells in peripheral blood of systemic lupus erythematosuss[J]. Journal of Peking University (Health Sciences), 2023, 55(6): 975-981.

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参考文献 32

1	Humbel M , Bellanger F , Fluder N , et al. Restoration of NK cell cytotoxic function with elotuzumab and daratumumab promotes elimination of circulating plasma cells in patients with SLE[J]. Front Immunol, 2021, 12, 645478. doi: 10.3389/fimmu.2021.645478
2	Cheng Q , Chen X , Xu J , et al. lncRNA X-inactive-specific transcript is a potential biomarker related to changes in CD4⁺T cell levels in systemic lupus erythematosus[J]. Rheumatol Autoimmun, 2022, 2 (3): 159- 174. doi: 10.1002/rai2.12048
3	Lin SJ , Kuo ML , Hsiao HS , et al. Cytotoxic function and cytokine production of natural killer cells and natural killer T-like cells in systemic lupus erythematosis regulation with interleukin-15[J]. Mediators Inflamm, 2019, 2019, 4236562.
4	Sordo-Bahamonde C , Lorenzo-Herrero S , Payer ÁR , et al. Mechanisms of apoptosis resistance to NK cell-mediated cytotoxicity in cancer[J]. Int J Mol Sci, 2020, 21 (10): 3726. doi: 10.3390/ijms21103726
5	Lorenzo-Herrero S , Sordo-Bahamonde C , Gonzalez S , et al. CD107a degranulation assay to evaluate immune cell antitumor activity[J]. Methods Mol Biol, 2019, 1884, 119- 130.
6	Myers JA , Miller JS . Exploring the NK cell platform for cancer immunotherapy[J]. Nat Rev Clin Oncol, 2021, 18 (2): 85- 100. doi: 10.1038/s41571-020-0426-7
7	Quatrini L , Della Chiesa M , Sivori S , et al. Human NK cells, their receptors and function[J]. Eur J Immunol, 2021, 51 (7): 1566- 1579. doi: 10.1002/eji.202049028
8	Kucuksezer UC , Aktas Cetin E , Esen F , et al. The role of natural killer cells in autoimmune diseases[J]. Front Immunol, 2021, 12, 622306. doi: 10.3389/fimmu.2021.622306
9	Bryceson YT , March ME , Barber DF , et al. Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells[J]. J Exp Med, 2005, 202 (7): 1001- 1012. doi: 10.1084/jem.20051143
10	Nielsen CM , White MJ , Goodier MR , et al. Functional significance of CD57 expression on human NK cells and relevance to disease[J]. Front Immunol, 2013, 4, 422.
11	Lu Z , Tian Y , Bai Z , et al. Increased oxidative stress contributes to impaired peripheral CD56^dimCD57⁺ NK cells from patients with systemic lupus erythematosus[J]. Arthritis Res Ther, 2022, 24 (1): 48. doi: 10.1186/s13075-022-02731-y
12	Banchereau R , Hong S , Cantarel B , et al. Personalized immunomonitoring uncovers molecular networks that stratify lupus patients[J]. Cell, 2016, 165 (3): 551- 565. doi: 10.1016/j.cell.2016.03.008
13	Li P , Jiang M , Li K , et al. Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity[J]. Nat Immunol, 2021, 22 (9): 1107- 1117. doi: 10.1038/s41590-021-00993-3
14	Furie R , Werth VP , Merola JF , et al. Monoclonal antibody targeting BDCA2 ameliorates skin lesions in systemic lupus erythematosus[J]. J Clin Invest, 2019, 129 (3): 1359- 1371. doi: 10.1172/JCI124466
15	Chatham WW , Furie R , Saxena A , et al. Long-term safety and efficacy of anifrolumab in adults with systemic lupus erythematosus: Results of a phase Ⅱ open-label extension study[J]. Arthritis Rheumatol, 2021, 73 (5): 816- 825. doi: 10.1002/art.41598
16	Kirou KA , Mavragani CP , Crow MK . Activation of type I inter-feron in systemic lupus erythematosus[J]. Expert Rev Clin Immunol, 2007, 3 (4): 579- 588. doi: 10.1586/1744666X.3.4.579
17	Le Bon A , Thompson C , Kamphuis E , et al. Cutting edge: Enhancement of antibody responses through direct stimulation of B and T cells by type I IFN[J]. J Immunol, 2006, 176 (4): 2074- 2078. doi: 10.4049/jimmunol.176.4.2074
18	Cucak H , Yrlid U , Reizis B , et al. Type Ⅰ interferon signaling in dendritic cells stimulates the development of lymph-node-resident T follicular helper cells[J]. Immunity, 2009, 31 (3): 491- 501. doi: 10.1016/j.immuni.2009.07.005
19	De Groof A , Ducreux J , Aleva F , et al. STAT3 phosphorylation mediates the stimulatory effects of interferon alpha on B cell dif-ferentiation and activation in SLE[J]. Rheumatology (Oxford), 2020, 59 (3): 668- 677.
20	Hochberg MC . Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus[J]. Arthritis Rheum, 1997, 40 (9): 1725.
21	Li H , Geng L , Cao Z , et al. CD56^brightCD16^- to CD57⁺CD56^dimCD16⁺ NK cell ratio discriminates disease activity and renal involvement in patients with systemic lupus erythematosus[J]. Clin Exp Rheumatol, 2023, 41 (9): 1768- 1776.
22	Yang Y , Day J , Souza-Fonseca Guimaraes F , et al. Natural killer cells in inflammatory autoimmune diseases[J]. Clin Transl Immunology, 2021, 10 (2): e1250. doi: 10.1002/cti2.1250
23	Streltsova MA , Erokhina SA , Kanevskiy LM , et al. Analysis of NK cell clones obtained using interleukin-2 and gene-modified K562 cells revealed the ability of "senescent" NK cells to lose CD57 expression and start expressing NKG2A[J]. PLoS One, 2018, 13 (12): e0208469. doi: 10.1371/journal.pone.0208469
24	Bahadorian D , Mollazadeh S , Mirazi H , et al. Regulatory NK cells in autoimmune disease[J]. Iran J Basic Med Sci, 2023, 26 (6): 609- 616.
25	Fresneda Alarcon M , McLaren Z , Wright HL . Neutrophils in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus: Same foe different M.O[J]. Front Immunol, 2021, 12, 649693. doi: 10.3389/fimmu.2021.649693
26	Shah D , Mahajan N , Sah S , et al. Oxidative stress and its biomarkers in systemic lupus erythematosus[J]. J Biomed Sci, 2014, 21 (1): 23. doi: 10.1186/1423-0127-21-23
27	Kennel KB , Greten FR . Immune cell-produced ROS and their impact on tumor growth and metastasis[J]. Redox Biol, 2021, 42, 101891. doi: 10.1016/j.redox.2021.101891
28	Song H , Park H , Kim YS , et al. L-kynurenine-induced apoptosis in human NK cells is mediated by reactive oxygen species[J]. Int Immunopharmacol, 2011, 11 (8): 932- 938. doi: 10.1016/j.intimp.2011.02.005
29	He H , Wang C , Liu G , et al. Isobavachalcone inhibits acute myeloid leukemia: Potential role for ROS-dependent mitochondrial apoptosis and differentiation[J]. Phytother Res, 2021, 35 (6): 3337- 3350. doi: 10.1002/ptr.7054
30	Bhattacharya A , Hegazy AN , Deigendesch N , et al. Superoxide dismutase 1 protects hepatocytes from type I interferon-driven oxidative damage[J]. Immunity, 2015, 43 (5): 974- 986. doi: 10.1016/j.immuni.2015.10.013
31	Tasdogan A , Kumar S , Allies G , et al. DNA damage-induced HSPC malfunction depends on ROS accumulation downstream of IFN-1 signaling and bid mobilization[J]. Cell Stem Cell, 2016, 19 (6): 752- 767. doi: 10.1016/j.stem.2016.08.007
32	Buang N , Tapeng L , Gray V , et al. Type Ⅰ interferons affect the metabolic fitness of CD8⁺ T cells from patients with systemic lupus erythematosus[J]. Nat Commun, 2021, 12 (1): 980. doi: 10.1038/s41467-021-21210-7

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