Natasha Vassileff, Jereme G. Spiers, Sarah E. Bamford, Rohan G. T. Lowe, Keshava K. Datta, Paul J. Pigram, Andrew F. Hill
Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.
神经炎症是神经退行性疾病的一个基本特征,通常出现在疾病病因的早期。小胶质细胞活化是神经炎症的一个重要诱因,可通过脂多糖(LPS)处理诱导小胶质细胞活化,导致一氧化氮合酶(iNOS)的诱导型表达,从而产生一氧化氮(NO)。一氧化氮通过 S-亚硝基化对半胱氨酸硫醇进行翻译后修饰,从而改变目标蛋白质的功能。此外,将这些经 NO 修饰的蛋白质包装到细胞外囊泡 (EVs) 中,可以在远处发出 NO 信号,从而进一步传播神经炎症表型。尽管如此,活化的小胶质细胞EVs的NO修饰蛋白质组尚未得到研究。本研究旨在确定 NO 信号在神经炎症中诱导的蛋白质翻译后修饰。从经 LPS 处理的小胶质细胞中分离出的 EVs 利用飞行时间-二次离子质谱(ToF-SIMS)进行质谱表面成像,此外还进行了碘标记和比较蛋白质组分析,以确定翻译后的 S-亚硝基化修饰。ToF-SIMS 成像成功鉴定了经 LPS 处理的小胶质细胞衍生 EV 蛋白中通过 NO 信号修饰的半胱氨酸硫醇侧链。此外,碘标记蛋白质组分析表明,经 LPS 处理的小胶质细胞 EVs 含有表明神经炎症的 S-亚硝基化蛋白质。这些蛋白包括已知的氮氧化物修饰蛋白和与 LPS 诱导的小胶质细胞活化相关的蛋白,它们可能在神经炎症交流中发挥重要作用。总之,这些结果表明,活化的小胶质细胞在神经炎症过程中可通过选择性包装 EVs 来产生广泛的 NO 信号变化。
{"title":"Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular vesicles","authors":"Natasha Vassileff, Jereme G. Spiers, Sarah E. Bamford, Rohan G. T. Lowe, Keshava K. Datta, Paul J. Pigram, Andrew F. Hill","doi":"10.1002/jev2.12455","DOIUrl":"10.1002/jev2.12455","url":null,"abstract":"<p>Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12455","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Carlos J Nogueras-Ortiz, Erden Eren, Pamela Yao, Elizabeth Calzada, Christopher Dunn, Olga Volpert, Francheska Delgado-Peraza, Maja Mustapic, Alexey Lyashkov, F Javier Rubio, Michael Vreones, Lesley Cheng, Yang You, Andrew F Hill, Tsuneya Ikezu, Erez Eitan, Edward J Goetzl, Dimitrios Kapogiannis
Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%–3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.
用 L1 细胞粘附分子(L1CAM)特异性抗体分离神经元衍生的细胞外囊泡(NDEVs)已被广泛用于鉴定中枢神经系统疾病的血液生物标记物。然而,方法论的全面验证需要在单个 NDEV 中证明 L1CAM,以及在来自其他细胞的单个 EV 中证明 L1CAM 水平较低或不存在。在这里,我们使用多种单个 EV 技术确定了神经元的来源,并确定了人体血液中 L1CAM 阳性 EV 的丰度。结果表明,L1CAM 外结构域的表位与神经元蛋白 β-III-tubulin、GAP43 和 VAMP2 共同表达在单个 EV 上,它们的水平随着 L1CAM 阳性 EV 的富集而增加。携带神经元蛋白 VAMP2 和 β-III-tubulin 的 L1CAM 阳性 EVs 含量为 30% 至 63%,而 L1CAM 阴性 EVs 含量为 0.8% 至 3.9%。血浆液相 L1CAM 不与单个 EV 结合。我们的研究结果支持将 L1CAM 作为分离血浆 NDEVs 的目标,并利用它们的货物来鉴定反映神经元功能的生物标记物。
{"title":"Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs","authors":"Carlos J Nogueras-Ortiz, Erden Eren, Pamela Yao, Elizabeth Calzada, Christopher Dunn, Olga Volpert, Francheska Delgado-Peraza, Maja Mustapic, Alexey Lyashkov, F Javier Rubio, Michael Vreones, Lesley Cheng, Yang You, Andrew F Hill, Tsuneya Ikezu, Erez Eitan, Edward J Goetzl, Dimitrios Kapogiannis","doi":"10.1002/jev2.12459","DOIUrl":"10.1002/jev2.12459","url":null,"abstract":"<p>Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%–3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12459","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vivian V. T. Nguyen, Joshua A. Welsh, Tobias Tertel, Andre Choo, Simonides I. van de Wakker, Kyra A. Y. Defourny, Bernd Giebel, Pieter Vader, Jayanthi Padmanabhan, Sai Kiang Lim, Esther N. M. Nolte-'t Hoen, Marianne C. Verhaar, R. Beklem Bostancioglu, Antje M. Zickler, Jia Mei Hong, Jennifer C. Jones, Samir EL Andaloussi, Bas W. M. van Balkom, André Görgens
Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.
{"title":"Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures","authors":"Vivian V. T. Nguyen, Joshua A. Welsh, Tobias Tertel, Andre Choo, Simonides I. van de Wakker, Kyra A. Y. Defourny, Bernd Giebel, Pieter Vader, Jayanthi Padmanabhan, Sai Kiang Lim, Esther N. M. Nolte-'t Hoen, Marianne C. Verhaar, R. Beklem Bostancioglu, Antje M. Zickler, Jia Mei Hong, Jennifer C. Jones, Samir EL Andaloussi, Bas W. M. van Balkom, André Görgens","doi":"10.1002/jev2.12463","DOIUrl":"10.1002/jev2.12463","url":null,"abstract":"<p>Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12463","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Irshad A. Sheikh, Monica T. Midura-Kiela, André Herchuelz, Sophie Sokolow, Pawel R. Kiela, Fayez K. Ghishan
Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3−/−) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, μCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3−/− mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.
{"title":"The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts","authors":"Irshad A. Sheikh, Monica T. Midura-Kiela, André Herchuelz, Sophie Sokolow, Pawel R. Kiela, Fayez K. Ghishan","doi":"10.1002/jev2.12450","DOIUrl":"10.1002/jev2.12450","url":null,"abstract":"<p>Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na<sup>+</sup>/Ca<sup>2+</sup> exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca<sup>2+</sup> transporter responsible for Ca<sup>2+</sup> extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca<sup>2+</sup> deposition due to reduced Ca<sup>2+</sup> entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3<sup>−/−</sup>) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, μCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3<sup>−/−</sup> mice, thus supporting the critical role of NCX3 in facilitating Ca<sup>2+</sup> uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12450","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141300747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rong Yang, Heng Zhang, Si Chen, Kaibin Lou, Meng Zhou, Mingchao Zhang, Rui Lu, Chunxia Zheng, Limin Li, Qihan Chen, Zhihong Liu, Ke Zen, Yanggang Yuan, Hongwei Liang
Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.
{"title":"Quantification of urinary podocyte-derived migrasomes for the diagnosis of kidney disease","authors":"Rong Yang, Heng Zhang, Si Chen, Kaibin Lou, Meng Zhou, Mingchao Zhang, Rui Lu, Chunxia Zheng, Limin Li, Qihan Chen, Zhihong Liu, Ke Zen, Yanggang Yuan, Hongwei Liang","doi":"10.1002/jev2.12460","DOIUrl":"10.1002/jev2.12460","url":null,"abstract":"<p>Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12460","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Giorgia Manni, Marco Gargaro, Doriana Ricciuti, Simona Fontana, Eleonora Padiglioni, Marco Cipolloni, Tommaso Mazza, Jessica Rosati, Alessandra di Veroli, Giulia Mencarelli, Benedetta Pieroni, Estevão Carlos Silva Barcelos, Giulia Scalisi, Francesco Sarnari, Alessandro di Michele, Luisa Pascucci, Francesca de Franco, Teresa Zelante, Cinzia Antognelli, Gabriele Cruciani, Vincenzo Nicola Talesa, Rita Romani, Francesca Fallarino
Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.
树突状细胞(DC)是免疫反应的重要协调者,也是自身免疫性疾病免疫调节的潜在靶点。人类羊水分泌物组富含免疫调节因子,其中细胞外囊泡 (EV) 是重要的组成部分。然而,这些EVs对树突状细胞亚群的影响仍有待探索。在这项研究中,我们调查了高度纯化的树突状细胞亚群与羊水干细胞系衍生的EVs(HAFSC-EVs)之间的相互作用。我们的研究结果表明,HAFSC-EVs 通过 CD29 受体介导的内化作用,优先被传统的 2 型树突状细胞(cDC2)吸收,从而形成一种以减少表达和产生促炎介质为特征的耐受性 DC 表型。此外,在共培养系统中用HAFSC-EV处理cDC2细胞后,表达调节性T细胞标记物Foxp3的T细胞比例高于用药物处理的对照细胞。此外,将经 HAFSC-EV 处理的 cDC2 移植到 EAE 小鼠模型中,可抑制自身免疫反应并改善临床症状。这些结果表明,HAFSC-EV 可以作为一种很有前途的工具,将炎症性 cDC2 重编程为耐受表型,并控制中枢神经系统的自身免疫反应,是研究 EV 在 DC 亚群中的作用的潜在平台。
{"title":"Amniotic fluid stem cell-derived extracellular vesicles educate type 2 conventional dendritic cells to rescue autoimmune disorders in a multiple sclerosis mouse model","authors":"Giorgia Manni, Marco Gargaro, Doriana Ricciuti, Simona Fontana, Eleonora Padiglioni, Marco Cipolloni, Tommaso Mazza, Jessica Rosati, Alessandra di Veroli, Giulia Mencarelli, Benedetta Pieroni, Estevão Carlos Silva Barcelos, Giulia Scalisi, Francesco Sarnari, Alessandro di Michele, Luisa Pascucci, Francesca de Franco, Teresa Zelante, Cinzia Antognelli, Gabriele Cruciani, Vincenzo Nicola Talesa, Rita Romani, Francesca Fallarino","doi":"10.1002/jev2.12446","DOIUrl":"10.1002/jev2.12446","url":null,"abstract":"<p>Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12446","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141283873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Renwei Jing, Leijie Zhang, Ruibin Li, Zhongqiu Yang, Jun Song, Qian Wang, Nan Cao, Gang Han, HaiFang Yin
Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EVVHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EVLV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EVLV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.
{"title":"Milk-derived extracellular vesicles functionalized with anti-tumour necrosis factor-α nanobody and anti-microbial peptide alleviate ulcerative colitis in mice","authors":"Renwei Jing, Leijie Zhang, Ruibin Li, Zhongqiu Yang, Jun Song, Qian Wang, Nan Cao, Gang Han, HaiFang Yin","doi":"10.1002/jev2.12462","DOIUrl":"10.1002/jev2.12462","url":null,"abstract":"<p>Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EV<sub>VHH</sub>) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EV<sub>LV</sub>), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EV<sub>LV</sub> effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 6","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12462","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141261998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Suraj Singh Rawat, Anand Kumar Keshri, Naina Arora, Rimanpreet Kaur, Amit Mishra, Rajiv Kumar, Amit Prasad
The excretory–secretory proteome plays a pivotal role in both intercellular communication during disease progression and immune escape mechanisms of various pathogens including cestode parasites like Taenia solium. The cysticerci of T. solium causes infection in the central nervous system known as neurocysticercosis (NCC), which affects a significant population in developing countries. Extracellular vesicles (EVs) are 30–150-nm-sized particles and constitute a significant part of the secretome. However, the role of EV in NCC pathogenesis remains undetermined. Here, for the first time, we report that EV from T. solium larvae is abundant in metabolites that can negatively regulate PI3K/AKT pathway, efficiently internalized by macrophages to induce AKT and mTOR degradation through auto-lysosomal route with a prominent increase in the ubiquitination of both proteins. This results in less ROS production and diminished bacterial killing capability among EV-treated macrophages. Due to this, both macro-autophagy and caspase-linked apoptosis are upregulated, with a reduction of the autophagy substrate sequestome 1. In summary, we report that T. solium EV from viable cysts attenuates the AKT–mTOR pathway thereby promoting apoptosis in macrophages, and this may exert immunosuppression during an early viable stage of the parasite in NCC, which is primarily asymptomatic. Further investigation on EV-mediated immune suppression revealed that the EV can protect the mice from DSS-induced colitis and improve colon architecture. These findings shed light on the previously unknown role of T. solium EV and the therapeutic role of their immune suppression potential.
排泄-分泌蛋白质组在疾病进展过程中的细胞间通信和各种病原体的免疫逃逸机制中都发挥着关键作用,其中包括像蛔虫这样的绦虫寄生虫。蛔虫的囊尾蚴会导致中枢神经系统感染,被称为神经囊尾蚴病(NCC),影响着发展中国家的大量人口。细胞外囊泡(EVs)是 30-150 纳米大小的颗粒,是分泌物的重要组成部分。然而,EV在NCC发病机制中的作用仍未确定。在这里,我们首次报道了蜱幼虫的EV富含能负向调节PI3K/AKT通路的代谢物,能被巨噬细胞有效内化,通过自身溶酶体途径诱导AKT和mTOR降解,同时这两种蛋白的泛素化显著增加。这导致经 EV 处理的巨噬细胞产生的 ROS 减少,杀死细菌的能力减弱。因此,巨噬细胞自噬和与 Caspase 链接的细胞凋亡都被上调,自噬底物 sequestome 1 减少。总之,我们报告说,来自存活囊肿的梭形芽孢杆菌 EV 可减弱 AKT-mTOR 通路,从而促进巨噬细胞的凋亡,这可能会在寄生虫在 NCC 中的早期存活阶段(主要是无症状阶段)发挥免疫抑制作用。对 EV 介导的免疫抑制的进一步研究发现,EV 可以保护小鼠免受 DSS 诱导的结肠炎的影响,并改善结肠结构。这些发现揭示了蜱EV之前未知的作用及其免疫抑制潜力的治疗作用。
{"title":"Taenia solium cysticerci's extracellular vesicles Attenuate the AKT/mTORC1 pathway for Alleviating DSS-induced colitis in a murine model","authors":"Suraj Singh Rawat, Anand Kumar Keshri, Naina Arora, Rimanpreet Kaur, Amit Mishra, Rajiv Kumar, Amit Prasad","doi":"10.1002/jev2.12448","DOIUrl":"10.1002/jev2.12448","url":null,"abstract":"<p>The excretory–secretory proteome plays a pivotal role in both intercellular communication during disease progression and immune escape mechanisms of various pathogens including cestode parasites like <i>Taenia solium</i>. The cysticerci of <i>T. solium</i> causes infection in the central nervous system known as neurocysticercosis (NCC), which affects a significant population in developing countries. Extracellular vesicles (EVs) are 30–150-nm-sized particles and constitute a significant part of the secretome. However, the role of EV in NCC pathogenesis remains undetermined. Here, for the first time, we report that EV from <i>T. solium</i> larvae is abundant in metabolites that can negatively regulate PI3K/AKT pathway, efficiently internalized by macrophages to induce AKT and mTOR degradation through auto-lysosomal route with a prominent increase in the ubiquitination of both proteins. This results in less ROS production and diminished bacterial killing capability among EV-treated macrophages. Due to this, both macro-autophagy and caspase-linked apoptosis are upregulated, with a reduction of the autophagy substrate sequestome 1. In summary, we report that <i>T. solium</i> EV from viable cysts attenuates the AKT–mTOR pathway thereby promoting apoptosis in macrophages, and this may exert immunosuppression during an early viable stage of the parasite in NCC, which is primarily asymptomatic. Further investigation on EV-mediated immune suppression revealed that the EV can protect the mice from DSS-induced colitis and improve colon architecture. These findings shed light on the previously unknown role of <i>T. solium</i> EV and the therapeutic role of their immune suppression potential.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 5","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12448","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parikh, Simon Spivack, Olivier Loudig
In the originally published article, author Kaushal Parikh's name was misspelled. This has been corrected in the online version of the article.
We apologize for this error.
Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parikh, Simon Spivack, Olivier Loudig在最初发表的文章中,作者Kaushal Parikh的名字拼错了。我们对此错误深表歉意。
{"title":"Correction to “Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed”","authors":"","doi":"10.1002/jev2.12453","DOIUrl":"10.1002/jev2.12453","url":null,"abstract":"<p>Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parikh, Simon Spivack, Olivier Loudig</p><p>In the originally published article, author Kaushal Parikh's name was misspelled. This has been corrected in the online version of the article.</p><p>We apologize for this error.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 5","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12453","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141071124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Marie Burt, Georgia Angelidou, Christopher Nils Mais, Christian Preußer, Timo Glatter, Thomas Heimerl, Rüdiger Groß, Javier Serrania, Gowtham Boosarpu, Elke Pogge von Strandmann, Janis A. Müller, Gert Bange, Anke Becker, Mareike Lehmann, Danny Jonigk, Lavinia Neubert, Hinrich Freitag, Nicole Paczia, Bernd Schmeck, Anna Lena Jung
The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae’s antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.
耐多药细菌病原体的不断出现对全球医疗保健构成了重大挑战,其中肺炎克雷伯氏菌是一个突出的威胁。我们对肺炎克雷伯菌的抗生素耐药机制进行了全面研究,重点是外膜囊泡和多粘菌素(一种最后的抗生素)。我们的研究表明,外膜囊泡能保护细菌免受多粘菌素的侵害。与未受多粘菌素影响的克雷伯氏菌的外膜囊泡相比,受多粘菌素 B(PB)影响的肺炎克雷伯氏菌的外膜囊泡由于囊泡化增加而显示出更强的保护效力。OMV 还能保护不同细菌科的细菌。我们使用精密切片肺片(PCLS)和鼠伤寒杆菌对这一点进行了体内外验证。在所有模型中,OMV 都能保护肺炎双球菌免受 PB 感染,并降低蛋白质水平上的相关应激反应。我们观察到 OMVs 的脂质组成在 PB 处理后发生了重大变化,从而影响了它们与 PB 的结合能力。肺炎克雷伯菌受到 PB 胁迫后,其单个 OMVs 的结合能力发生了改变,这可能与其释放的囊泡中的脂质 A 数量减少有关。虽然每个囊泡的脂质 A 量减少了,但由于脂质 A 的总和增加了,因此 PB 结合位点也增加了,囊泡数量的总体增加导致了保护能力的增强。这就揭示了与对照 OMV 相比,肺炎克氏菌受 PB 胁迫的 OMV 对 PB 的保护效力发生改变的机制。利用人工囊泡在体外证实了脂质 A 依赖性对 PB 的保护作用。此外,人工囊泡在体内外都成功地保护了克雷伯氏菌免受肺炎双球菌的感染。研究结果表明,OMV 可通过与多粘菌素结合,有效地充当诱饵,阻止抗生素与细胞表面的相互作用,从而起到保护细菌的作用。我们的研究结果为了解抗生素交叉保护的机制提供了宝贵的见解,并为开发新型治疗干预措施提供了潜在的途径,以应对不断升级的耐多药细菌感染的威胁。
{"title":"Lipid A in outer membrane vesicles shields bacteria from polymyxins","authors":"Marie Burt, Georgia Angelidou, Christopher Nils Mais, Christian Preußer, Timo Glatter, Thomas Heimerl, Rüdiger Groß, Javier Serrania, Gowtham Boosarpu, Elke Pogge von Strandmann, Janis A. Müller, Gert Bange, Anke Becker, Mareike Lehmann, Danny Jonigk, Lavinia Neubert, Hinrich Freitag, Nicole Paczia, Bernd Schmeck, Anna Lena Jung","doi":"10.1002/jev2.12447","DOIUrl":"10.1002/jev2.12447","url":null,"abstract":"<p>The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with <i>Klebsiella pneumoniae</i> being a prominent threat. We conducted a comprehensive study on <i>K. pneumoniae</i>’s antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed <i>K. pneumoniae</i> exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed <i>Klebsiella</i>. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and <i>Galleria mellonella</i>. In all models, OMVs protected <i>K. pneumoniae</i> from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed <i>K. pneumoniae</i> could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed <i>K. pneumoniae</i> compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected <i>Klebsiella</i> from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 5","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12447","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141065752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}