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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Hickman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Wildfires adversely impact air quality and public health worldwide. Exposures to wildfire smoke are linked to adverse health outcomes, including cardiopulmonary diseases. Critical research gaps remain surrounding the underlying biological pathways leading to wildfire-induced health effects. The regulation of intercellular communication and downstream toxicity driven by extracellular vesicles (EVs) is an important, understudied biological mechanism. This study investigated EVs following a wildfire smoke-relevant in vitro exposure. We hypothesized that woodsmoke (WS) would alter the proteomic content of EVs secreted in organotypic in vitro airway models. Exposures were carried out using a tri-culture model of alveolar epithelial cells, fibroblasts, and endothelial cells and a simplified co-culture model of alveolar epithelial cells and fibroblasts to inform responses across different cell populations. Epithelial cells were exposed to WS condensate and EVs were isolated from basolateral conditioned medium following 24 h exposure. WS exposure did not influence EV particle characteristics, and it moderately increased EV count. Exposure caused the differential loading of 25 and 35 proteins within EVs collected from the tri- and co-culture model, respectively. EV proteins involved in extracellular matrix degradation and wound healing were consistently modulated across both models. However, distinct proteins involved in the wound healing pathway were altered between models, suggesting unique but concerted efforts across cell types to communicate in response to injury. These findings demonstrate that a wildfire-relevant exposure alters the EV proteome and suggest an impact on EV-mediated intercellular communication. Overall, results demonstrate the viability of organotypic approaches in evaluating EVs to investigate exposure-induced biomarkers and underlying mechanisms. Findings also highlight the impact of differences in the biological complexity of in vitro models used to evaluate the effects of inhaled toxicants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute radiation syndrome (ARS) develops after exposure to high doses of ionizing radiation and features immune suppression and organ failure. Currently, there are no diagnostics to identify the occurrence or severity of exposure and there are limited treatments and preventative strategies to mitigate ARS. Extracellular vesicles (EVs) are mediators of intercellular communication that contribute to immune dysfunction across many diseases. We investigated if EV cargo can identify whole body irradiation (WBIR) exposure and if EVs promote ARS immune dysfunction. We hypothesized that beneficial EVs derived from mesenchymal stem cells (MSC-EVs) would blunt ARS immune dysfunction and might serve as prophylactic radioprotectants. Mice received WBIR (2 or 9 Gy) with assessment of EVs at 3 and 7 days after exposure. LC-MS/MS proteomic analysis of WBIR-EVs found dose-related changes as well as candidate proteins that were increased with both doses and timepoints (34 total) such as Thromboxane-A Synthase and lymphocyte cytosolic protein 2. Suprabasin and Sarcalumenin were increased only after 9 Gy suggesting these proteins may indicate high dose/lethal exposure. Analysis of EV miRNAs identified miR-376 and miR-136, which were increased up to 200- and 60-fold respectively by both doses of WBIR and select miRNAs such as miR-1839 and miR-664 were increased only with 9 Gy. WBIR-EVs (9 Gy) were biologically active and blunted immune responses to LPS in RAW264.7 macrophages, inhibiting canonical signaling pathways associated with wound healing and phagosome formation. When given 3 days after exposure, MSC-EVs slightly modified immune gene expression changes in the spleens of mice in response to WBIR and in a combined radiation plus burn injury exposure (RCI). MSC-EVs normalized the expression of certain key immune genes such as NFκBia and Cxcr4 (WBIR), Map4k1 , Ccr9 and Cxcl12 (RCI) and lowered plasma TNFα cytokine levels after RCI. When given prophylactically (24 and 3 hours before exposure), MSC-EVs prolonged survival to the 9 Gy lethal exposure. Thus, EVs are important participants in ARS. EV cargo might be used to diagnose WBIR exposure, and MSC-EVs might serve as radioprotectants to blunt the impact of toxic radiation exposure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Seim, Herring, Mordant, Willis, Wallet, Coleman and Maile.)

Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (<72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.