唾液链球菌胞外囊泡通过活化自然杀伤细胞加剧系统性红斑狼疮肾炎病理进展

doi:10.12122/j.issn.1673-4254.2026.03.08

摘要/Abstract

摘要：

目的探讨唾液链球菌来源胞外囊泡（SS-EVs）对自然杀伤（NK）细胞活化功能的调控作用及其在系统性红斑狼疮（SLE）疾病进展中的潜在机制。方法收集35例SLE患者及38例健康对照者的临床样本，检测其外周血T细胞、B细胞及NK细胞的数量与比例。提取患者粪便DNA，通过PCR扩增出唾液链球菌DNA片段，并通过琼脂糖凝胶电泳评估其丰度水平。采用超速离心技术分离SS-EVs，并通过透射电镜、纳米颗粒追踪分析和Western blotting对其进行表征。将SS-EVs与NK细胞共培养，通过定量聚合酶链式反应（qPCR）及流式细胞术体外检测NK细胞的活化状态及其细胞毒功能；将24只MRL/lpr小鼠随机分成3组，8只/组，每次分别灌胃100 μL PBS、100 μL含20 μg的E.coli-EVs和SS-EVs，通过HE染色、免疫荧光及ELISA评估肾脏病理改变、免疫复合物沉积及外周炎症因子水平，从而评估SS-EVs对狼疮性肾炎的影响。结果 SLE患者外周血中NK细胞数量与比例下降（P<0.0001），粪便中SS丰度升高，且与SLEDAI评分呈正相关（R²=0.8369）。体外研究显示，SS-EVs上调NK细胞表面激活性受体的表达（P<0.01），增强其对靶细胞的杀伤效能；体内实验发现，SS-EVs可加剧小鼠肾组织炎症反应，促进C3和IgG免疫复合物沉积，升高血清中IL-6、TNF-α、IL-8及CCL20等炎症因子水平（P<0.05）。结论 SLE患者的肠道中唾液链球菌富集，并通过释放SS-EVs促进NK细胞毒性功能的增强，诱导肾脏炎症反应及免疫复合物沉积，从而加重SLE的疾病进展。SS及其胞外囊泡可能为SLE的防治提供全新的靶向策略。

关键词: 系统性红斑狼疮, 唾液链球菌, 胞外囊泡, 自然杀伤细胞, 免疫调节

Abstract:

Objective To investigate the regulatory effects of Streptococcus salivarius-derived extracellular vesicles (SS-EVs) on natural killer (NK) cell activation and cytotoxic function and their role in progression of systemic lupus erythematosus (SLE). Methods Peripheral blood and fecal samples were collected from 35 SLE patients and 38 healthy individuals to assess the number and proportion of peripheral T cells, B cells, and NK cells. Fecal DNA was extracted for PCR amplification of S. salivarius DNA fragments and quantitative analysis using agarose gel electrophoresis. SS-EVs isolated by ultracentrifugation and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting were co-cultured with NK cells, whose activation status and cytotoxicity were evaluated in vitro using qPCR and flow cytometry. Twenty-four MRL/lpr mice were randomized into 3 groups (n=8) for treatment with gavage of 100 μL PBS or 20 μg E. coli-EVs or SS-EVs in 100 μL PBS. Kidney pathology, immune complex deposition, and peripheral cytokine levels were assessed with HE staining, immunofluorescence staining, and ELISA. Results The number and proportion of peripheral NK cells were significantly reduced (P<0.0001) and the fecal abundance of S. salivarius was markedly increased in positive correlation with SLEDAI scores (R²=0.8369) in SLE patients. In vitro studies showed that SS-EVs significantly upregulated NK cell-activating receptors and enhanced NK-mediated cytotoxicity. In MRL/lpr mice, SS-EV treatment markedly exacerbated renal inflammation, promoted C3 and IgG immune complex deposition, and significantly increased serum levels of IL-6, TNF-α, IL-8, and CCL20. Conclusion S. salivarius is enriched in the gut of SLE patients and promotes NK-cell activation by releasing SS-EVs, which induce renal inflammation and immune complex deposition and contributes to SLE progression. S. salivarius and its EVs may represent novel therapeutic targets for treatment of SLE.

Key words: systemic lupus erythematosus, Streptococcus salivarius, extracellular vesicles, natural killer cells, immunomodulation

肖洁, 金凌岳, 段丽娜, 龚英, 李海侠. 唾液链球菌胞外囊泡通过活化自然杀伤细胞加剧系统性红斑狼疮肾炎病理进展[J]. 南方医科大学学报, 2026, 46(3): 541-549.

Jie XIAO, Lingyue JIN, Lina DUAN, Ying GONG, Haixia LI. Streptococcus salivarius-derived extracellular vesicles exacerbate progression of lupus nephritis by activating natural killer cells[J]. Journal of Southern Medical University, 2026, 46(3): 541-549.

图/表 7

表1 引物序列

Tab.1 Primer sequences for qPCR

Primer name	Sequence (5'-3')
GAPDH -FOR	CTGTTCGACAGTCAGCCGCATC
GAPDH -REV	GCGCCCAATACGACCAAATCCG
TNF-FOR	GAGGCCAAGCCCTGGTATG
TNF-REV	CGGGCCGATTGATCTCAGC
GZMB-FOR	CCCTGGGAAAACACTCACACA
GZMB-REV	GCACAACTCAATGGTACTGTCG
IL2-FOR	AACTCCTGTCTTGCATTGCAC
IL2-REV	GCTCCAGTTGTAGCTGTGTTT
Perforin-FOR	GACTGCCTGACTGTCGAGG
Perforin-REV	TCCCGGTAGGTTTGGTGGAA
NKG2D-FOR	CCTTGACCGAAAGTTACTGTGG
NKG2D-REV	GGCTGGCATTTTGAGACATACAA
NKG2A-FOR	AGCTCCATTTTAGCAACTGAACA
NKG2A-REV	CAACTATCGTTACCACAGAGGC

表2 SLE组与HC组的临床特征

Tab.2 Clinical characteristics of the SLE patients and healthy control subjects (Mean±SD)

General information		SLE group (n=35)	HC group (n=38)	P
Gender [n (%)]	Female	30 (85.71%)	31 (81.58%)	0.756
Gender [n (%)]	Male	5 (14.29%)	7 (18.42%)	0.756
Age (year)		36.57±12.50	39.34±11.84	0.334
WBC (10⁹/L)		6.07±2.81	5.66±1.17	0.422
LYM (10⁹/L)		1.53±0.76	2.20±0.47	<0.001
NEU (10⁹/L)		4.12±2.09	3.12±0.93	0.043
RBC (10¹²/L)		4.12±0.67	4.89±0.32	<0.001
HGB (g/L)		113.48±14.73	135.67±17.63	<0.001
HCT (L/L)		0.37±0.38	0.42±0.43	<0.001
PLT (10⁹/L)		243.42±82.22	255.64±59.45	0.512
SLEDAI		6.74±3.35	-	-
WBC: White blood cell; LYM: Lymphocyte; NEU: Neutrophil; RBC: Red blood cell; HGB: Hemoglobin; HCT: Hematocrit; PLT: Platelet. Data are presented as Mean±SD. P values were calculated by Mann-Whitney U test or t test.

图1 粪便SS核酸琼脂糖凝胶电泳结果

Fig.1 Agarose gel electrophoresis of fecal Streptococcus salivarius (SS) nucleic acids. A: Results of agarose gel electrophoresis. Lane 1: Deionized water (negative control); Lane 2: SS standard strain (positive control). a: Lanes 3-12 are SLE group, and lanes 13-24 are HC group; b: Lanes 3-10 are SLE group, and lanes 11-22 are HC group; c: Lanes 3-11 are SLE group, and lanes 12-17 are HC group; d: Lanes 3-10 are SLE group, and lanes 11-18 are HC group. B: Correlation analysis between the grayscale value of fecal SS nucleic acids from SLE patients and the SLEDAI score.

图2 SLE患者外周血免疫细胞亚群分布情况

Fig.2 Distribution of peripheral blood immune cell subsets in SLE patients. A, B: NK cell proportions and absolute counts in SLE patients and HC groups. C, D: Total T cell absolute counts and subset proportions. E: CD8+ T cell subset proportions. F: CD4+ T cell absolute counts (n=35 in SLE group, n=38 in HC group). *P<0.05, ***P<0.001, ****P<0.0001.

图3 SS-EVs的物理特性及NK细胞受体调控作用

Fig.3 Physical properties of Streptococcus salivarius-derived extracellular vesicles (SS-EVs)and their regulatory effect on NK cell receptors. A: Transmission electron microscopy (TEM) of SS-EVs (Scale bar=100 nm). B: NTA analysis showing size distribution of SS-EVs (100-200 nm). C: Western blotting of LTA protein expression in SS-EVs. D: qPCR analysis of mRNA expression changes of the activating receptors (TNF, GZMB, NKG2D, IL-2, and Perforin) and inhibitory receptors (NKG2A) in NK cells co-cultured with SS-EVs (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图4 SS-EVs增强NK细胞对靶细胞的毒性作用

Fig.4 SS-EVs enhance cytotoxic effect of NK cells on target cells. A-D: Relationship between NK cell to target cell ratio (1:1 to 4:1) and NK cell cytotoxicity at a fixed SS-EV concentration (1 μg/mL) (n=3). E-H: Flow cytometry analysis of the effects of varying SS-EV concentrations (0.1-10 μg/mL) on NK cell cytotoxicity against 4 target cells (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图5 SS-EVs灌胃加重MRL/lpr小鼠肾脏病理损伤

Fig.5 SS-EVs gavage aggravates kidney pathological damage in MRL/lpr mice. A: Schematic diagram of animal grouping and gavage. B: Significant inflammatory cell infiltration in the kidneys of mice gavaged with SS-EVs. C: Significant perivascular inflammatory cell infiltration in the kidneys of mice gavaged with SS-EVs. D: SS-EVs damage the renal medulla and promote immune complex deposition. E:SS-EVs induce NK cell infiltration in the kidneys and renal apoptosis. F-I: ELISA for IL-6, TNF, IL-8, and CCL20 levels in mouse serum (n=6). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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