Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet‐rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro‐inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin α_IIbβ₃, while binding also occurred in a CX3CL1–CX3CR1‐dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40‐ and P‐selectin interactions. Flow‐resistant monocyte adhesion to platelet EV‐treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin α_IIbβ₃ and P‐selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro‐inflammatory phenotype, stimulating vascular remodelling.

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell’s redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.

Nontypeable Haemophilus influenzae (NTHI), a common colonizer of lungs of patients with chronic obstructive pulmonary disease (COPD), can enhance expression of the cellular receptor intercellular adhesion molecule 1 (ICAM‐1), which in turn can be used by major group human rhinoviruses (HRVs) for attachment. Here, we evaluated the effect of NTHI‐induced up‐regulation of ICAM‐1 on viral replication and inflammatory responses toward different respiratory viruses. Therefore, human bronchial epithelial cells were pretreated with heat‐inactivated NTHI (hi‐NTHI) and subsequently infected with either HRV16 (major group), HRV1B (minor group), or respiratory syncytial virus (RSV). Pretreatment with hi‐NTHI significantly up‐regulated ICAM‐1 in BEAS‐2B cells and primary bronchial epithelial cells. Concomitantly, release of infectious HRV16 particles was increased in cells pretreated with hi‐NTHI. Pretreatment with hi‐NTHI also caused a significant increase in HRV16 RNA, whereas replication of HRV1B and RSV were increased to a far lesser extent and only at later time points. Interestingly, release of IL‐6 and IL‐8 after RSV, but not HRV, infection was synergistically increased in hi‐NTHI‐pretreated BEAS 2B cells. In summary, exposure to hi‐NTHI significantly enhanced sensitivity toward HRV16 but not HRV1B or RSV, probably through ICAM‐1 up‐regulation. Furthermore, hi‐NTHI pretreatment may enhance the inflammatory response to RSV infection, suggesting that preexisting bacterial infections might exaggerate inflammation during secondary viral infection.—Gulraiz, F., Bellinghausen, C., Bruggeman, C. A., Stassen, F. R., Haemophilus influenzae increases the susceptibility and inflammatory response of airway epithelial cells to viral infections. FASEB J. 29, 849–858 (2015). www.fasebj.org