Immunoaffinity-enriched salivary small extracellular vesicles in periodontitis

Chun Liu, C. Seneviratne, Carlos Palma, Greg Rice, Carlos Salomon, Ramin Khanabdali, S. Ivanovski, Pingping Han
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Abstract

Aim: Saliva extracellular vesicles (EVs) serve as a significant reservoir of biomarkers that may be of clinical use in disease diagnosis. Saliva, however, contains EVs of both host- and bacterial- origin. Identifying suitable EVs for disease diagnosis involves enriching host EVs and limiting non-host contamination with effective isolation methods. The objectives of this research were: (1) to evaluate the salivary EVs enrichment in 12 periodontally healthy patients by two different methods: size exclusion chromatography (SEC) and bead-based immunoaffinity capture (EXO-NET®); (2) to analyze the variance expression of inflammatory cytokines in EXO-NET-enriched EVs, comparing individuals with periodontitis (n = 20) to non-periodontitis (n = 12). Methods: Whole unstimulated saliva samples were collected from 12 periodontally healthy and 20 periodontitis patients. EVs were isolated from the 12 non-periodontitis patients using SEC (referred to as SEC-EVs) and EXO-NET (referred to as EXO-NET EVs), after which their total protein content, 37 EV surface markers, and bacterial pathogens expression were compared. Subsequently, the inflammatory cytokines expression levels (interleukin-IL-6, IL-1β, IL-8, and IL-10) in EXO-NET EVs were measured for non-periodontitis and periodontitis. Results: EXO-NET EVs contained more EV-specific protein and substantially higher expression of EV surface markers (CD9, CD81, CD63), but less pathogenic DNA was detected compared to that in SEC-EVs. Additionally, EXO-NET EVs from periodontitis patients contained higher amounts of IL-6 and IL-8, and decreased IL-10, compared to those from non-periodontitis patients. Conclusion: The findings suggest that immunoaffinity capture (EXO-NET) is a dependable method for salivary EVs enrichment, resulting in a higher yield of host EVs with reduced bacterial DNA detection compared to SEC. Furthermore, the research proposes that immunoaffinity capture enriched EVs can function as biomarkers for periodontitis, demonstrated by an increased expression of proinflammatory cytokines from periodontitis patients.
牙周炎中富含免疫亲和力的唾液小细胞外囊泡
目的:唾液细胞外囊泡(EVs)是重要的生物标志物库,可用于临床疾病诊断。然而,唾液中既含有来源于宿主的EVs,也含有来源于细菌的EVs。要鉴别出适用于疾病诊断的 EVs,需要用有效的分离方法富集宿主 EVs 并限制非宿主污染。本研究的目标是(1)通过两种不同的方法:尺寸排阻色谱法(SEC)和珠式免疫亲和捕获法(EXO-NET®),评估12名牙周健康患者唾液EVs的富集情况;(2)比较牙周炎患者(n = 20)和非牙周炎患者(n = 12),分析EXO-NET富集的EVs中炎症细胞因子的表达差异。 研究方法从 12 名牙周健康的患者和 20 名牙周炎患者身上采集未经刺激的唾液样本。使用 SEC(简称 SEC-EVs)和 EXO-NET(简称 EXO-NET EVs)从 12 名非牙周炎患者的唾液中分离出 EVs,然后比较其总蛋白含量、37 种 EV 表面标记物和细菌病原体的表达。随后,测量了非牙周炎和牙周炎EXO-NET EVs中炎症细胞因子(白细胞介素-IL-6、IL-1β、IL-8和IL-10)的表达水平。 结果显示EXO-NET EVs含有更多的EV特异性蛋白,EV表面标记物(CD9、CD81、CD63)的表达量也大幅提高,但与SEC-EVs相比,检测到的病原体DNA较少。此外,与非牙周炎患者的EXO-NET EVs相比,牙周炎患者的EXO-NET EVs含有更多的IL-6和IL-8,而IL-10含量较低。 结论研究结果表明,免疫亲和捕获(EXO-NET)是一种可靠的唾液EVs富集方法,与SEC相比,它能获得更多的宿主EVs,同时减少细菌DNA的检测。此外,研究还提出,免疫亲和捕获富集的 EVs 可作为牙周炎的生物标记物,牙周炎患者促炎细胞因子的表达增加就是证明。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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