Human milk extracellular vesicles preserve bronchial epithelial barrier integrity and reduce TLR3-induced inflammation <i>in vitro</i>

Breast milk is essential for facilitating the growth and development of infants and for providing immune protection against viral infections in the infant’s airways. Yet, regulation of inflammation by milk components may be needed to reduce immune pathology. While milk-derived extracellular vesicles (EVs) are bestowed with immunomodulatory capacities, their role in bronchial epithelial barrier function and inflammation has not yet been examined. We hypothesised that during feeding, milk is not only ingested, but aerosols containing milk EVs are inhaled and locally delivered to the infant’s airways to suppress aberrant inflammation. A bronchial epithelial model of viral infection was used to explore the direct effect of milk EVs on cellular barrier function and cytokine release during stimulation with a viral dsRNA analogue (Poly I:C). We demonstrate that milk EVs improved the dsRNA-mediated decrease in ionic barrier integrity, limited tight junction reorganisation and reduced inflammatory cytokine production (IL-6, IL-8 and TNF-α). This protective response was EV-mediated, could be successfully titrated and exhibited a time-dependent response. The results indicate that if EV-containing milk aerosols were to be inhaled during feeding, this may lead to protection of the airway integrity from adverse inflammatory effects

Surface protein profiling of milk and serum extracellular vesicles unveil body fluid and cell-type signatures and insights on vesicle biogenesis

The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin

Surface protein profiling of milk and serum extracellular vesicles unveil body fluid and cell-type signatures and insights on vesicle biogenesis

The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin

Extracellular vesicles in joint disease and therapy

The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and their linking medium, the synovial fluid, in particular. Further insight is urgently needed in order to appreciate the full potential of EVs and to exploit these in EV-mediated therapies. Physiologic joint homeostasis is a prerequisite for proper functioning of joints and we postulate that EVs play a key role in the regulation of joint homeostasis and hence can have an important function in re-establishing disturbed joint homeostasis, and, in parallel, in the regeneration of articular tissues. In this mini-review EVs in the joint are explained from a historical perspective in both health and disease, including the potential niche for EVs in articular tissue regeneration. Furthermore, the translational potential of equine models for human joint biology is discussed. Finally, the use of MSC-derived EVs that is recently gaining ground is highlighted and recommendations are given for further EV research in this field

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges

Proteome and phospholipidome interrelationship of synovial fluid-derived extracellular vesicles in equine osteoarthritis: An exploratory ‘multi-omics’ study to identify composite biomarkers

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease