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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (6): 975-981. doi: 10.19723/j.issn.1671-167X.2023.06.004

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Interferon-α mediating the functional damage of CD56dimCD57+natural killer cells in peripheral blood of systemic lupus erythematosuss

Xiang-ge ZHAO,Jia-qing LIU,Hui-na HUANG,Zhi-min LU,Zi-ran BAI,Xia LI,Jing-jing QI*()   

  1. Department of Immunology, College of Basic Medical Science, Dalian Medical University, Dalian 116044, Liaoning, China
  • Received:2023-07-06 Online:2023-12-18 Published:2023-12-11
  • Contact: Jing-jing QI E-mail:jingjingqi_nju@126.com
  • Supported by:
    the National Natural Science Foundation of China(82201990);the National Natural Science Foundation of China(82071834);the National Natural Science Foundation of China(82271839);Science and Technology Talent Innovation Support Project of Dalian(2022RQ070)

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Abstract:

Objective: To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ 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. CD56dimCD57+ 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 CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ 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 CD56dimCD57+ NK killing function.

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

CLC Number: 

  • R593.2

Figure 1

Expression level of total NK cell killing functional molecules in peripheral blood of SLE patients HC, healthy control; SLE, systemic lupus erythematosus; NK, natural killer."

Figure 2

Comparison of cytotoxic function of peripheral blood CD56dimCD57+ NK cell to kill K562 cells between HC and SLE patients 7-AAD, 7-amino-actinomycin D; HC, healthy control; SLE, systemic lupus erythematosus; NK, natural killer. **P < 0.01."

Figure 3

Effects of H2O2and NAC on PRF expression in CD56dimCD57+ NK cell A, expression of perforin in CD56dimCD57+NK cells after peripheral blood PBMC of HC were exposed to different concentrations of H2O2 (20, 40 and 80 μmol/L) for 24 h. *P < 0.05; **P < 0.01. B, expression of perforin in CD56dimCD57+NK cells cultured with NAC (5 mmol/L) in peripheral blood of SLE patients for 24 h.**P < 0.01. NAC, N-acetyl cysteine; NK, natural killer; PBMC, peripheral blood mononuclear cell; H2O2, hydrogen peroxide; HC, healthy control; SLE, systemic lupus erythematosus; PRF, perforin."

Figure 4

Level of IFN-α and expression of IFNAR on CD56dimCD57+ NK cell in peripheral blood of HC and SLE patients A, IFN-α levels in peripheral blood serum of HC and SLE; ***P < 0.001. B, the expression level of IFNAR1 on the surface of CD56dimCD57+NK cell in peripheral blood of HC and SLE; *P < 0.05. C, the expression level of IFNAR2 on the surface of CD56dimCD57+NK cell in peripheral blood of HC and SLE. *P < 0.05; IFN-α, interferon-α; SLEDAI, systemic lupus erythematosus disease activity index; HC, healthy control; SLE, systemic lupus erythematosus; IFNAR, interferon-α receptor; NK, natural killer; MFI, mean fluorescence intensity."

Figure 5

Effects of IFN-α on apoptosis and mtROS production of CD56dimCD57+NK cells A, peripheral blood NK cells of HC were sorted and stimulated with IFN-α (1 000 U/mL) for 72 h to detect apoptosis of CD56dimCD57+NK cells, *P < 0.05; B, peripheral blood NK cells of HC were sorted and stimulated with IFN-α (1 000 U/mL) for 24, 48 and 72 h, mtROS expression in CD56dimCD57+NK cells was detected by flow cytometry, *P < 0.05, ****P < 0.000 1.7-AAD, 7-amino-actinomycin D; IFN-α, interferon-α; MFI, mean fluorescence intensity; mtROS, mitochondrial reactive oxygen species; NK, natural killer; HC, healthy control."

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