Hickman Elise, Carberry Victoria, Carberry Celeste, Cooper Bethanie, Mordant Angie L, Mills Allie, Sokolsky Marina, Herring Laura E, Alexis Neil E, Rebuli Meghan E, Jaspers Ilona, Sheats Katie, Rager Julia E
Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
PLoS One. 2025 Jan 24;20(1):e0315743. doi: 10.1371/journal.pone.0315743. eCollection 2025.
Growing evidence supports the importance of extracellular vesicle (EV) as mediators of communication in pathological processes, including those underlying respiratory disease. However, establishing methods for isolating and characterizing EVs remains challenging, particularly for respiratory samples. This study set out to address this challenge by comparing different EV isolation methods and evaluating their impacts on EV yield, markers of purity, and proteomic signatures, utilizing equine/horse bronchoalveolar lavage samples. Horses can serve as effective translational animal models for respiratory studies due to similarities with human immune responses, shared environmental exposures, and naturally occurring respiratory diseases including asthma. Further, horses are long-lived large animals that allow for longitudinal sample collection, and provide large sample volume and cell yield, which are particularly useful since EV research is commonly limited by low sample yields. Here, EVs were isolated from horse bronchoalveolar lavage fluid (BALF) using four different methods (ultracentrifugation, microcentrifugation, and two sizes of size exclusion chromatography columns) and characterized by measuring particle counts, EV purity, total protein yield, and proteomic cargo, with a specific focus on vesicle surface marker expression potentially informing cell type of origin. We found that size exclusion chromatography yielded the highest particle counts, greatest EV purity markers and elevated vesicle surface marker expression. Overall proteomic profiles differed across isolation methods, with size exclusion chromatography clustering separately from centrifugation. Taken together, our results demonstrate that different isolation methods impact characteristics of EVs, notably that size exclusion chromatography, compared to centrifugation methods, resulted in higher EV purity and better characterized proteomic diversity, including information on EV cell-of-origin. This is the first study to characterize proteomic profiles of EVs following different isolation methods using equine BALF. The results of this study will pave the way for future studies using equine and human samples to characterize respiratory tract EVs.
越来越多的证据支持细胞外囊泡（EV）在病理过程（包括呼吸系统疾病的潜在病理过程）中作为通讯介质的重要性。然而，建立分离和表征EV的方法仍然具有挑战性，特别是对于呼吸道样本。本研究旨在通过比较不同的EV分离方法，并评估它们对EV产量、纯度标志物和蛋白质组特征的影响，利用马的支气管肺泡灌洗样本解决这一挑战。由于马与人类免疫反应相似、有共同的环境暴露以及存在包括哮喘在内的自然发生的呼吸系统疾病，马可以作为呼吸系统研究有效的转化动物模型。此外，马是长寿的大型动物，允许进行纵向样本采集，并提供大量样本体积和细胞产量，这特别有用，因为EV研究通常受到低样本产量的限制。在这里，使用四种不同方法（超速离心、微量离心和两种尺寸的尺寸排阻色谱柱）从马支气管肺泡灌洗液（BALF）中分离EV，并通过测量颗粒计数、EV纯度、总蛋白产量和蛋白质组成分进行表征，特别关注可能指示起源细胞类型的囊泡表面标志物表达。我们发现尺寸排阻色谱法产生的颗粒计数最高、EV纯度标志物最显著且囊泡表面标志物表达升高。不同分离方法的总体蛋白质组谱不同，尺寸排阻色谱法与离心法分开聚类。综上所述，我们的结果表明不同的分离方法会影响EV的特征，特别是与离心法相比，尺寸排阻色谱法可产生更高的EV纯度和更具特征的蛋白质组多样性，包括有关EV起源细胞的信息。这是第一项使用马BALF对不同分离方法后的EV蛋白质组谱进行表征的研究。本研究结果将为未来使用马和人类样本表征呼吸道EV的研究铺平道路。
