In vivo Kinetic Biodistribution of Nano‐Sized Outer Membrane Vesicles Derived from Bacteria

Evaluation of kinetic distribution and behaviors of nanoparticles in vivo provides crucial clues into their roles in living organisms. Extracellular vesicles are evolutionary conserved nanoparticles, known to play important biological functions in intercellular, inter‐species, and inter‐kingdom communication. In this study, the first kinetic analysis of the biodistribution of outer membrane vesicles (OMVs)—bacterial extracellular vesicles—with immune‐modulatory functions is performed. OMVs, injected intraperitoneally, spread to the whole mouse body and accumulate in the liver, lung, spleen, and kidney within 3 h of administration. As an early systemic inflammation response, increased levels of TNF‐α and IL‐6 are observed in serum and bronchoalveolar lavage fluid. In addition, the number of leukocytes and platelets in the blood is decreased. OMVs and cytokine concentrations, as well as body temperature are gradually decreased 6 h after OMV injection, in concomitance with the formation of eye exudates, and of an increase in ICAM‐1 levels in the lung. Following OMV elimination, most of the inflammatory signs are reverted, 12 h post‐injection. However, leukocytes in bronchoalveolar lavage fluid are increased as a late reaction. Taken together, these results suggest that OMVs are effective mediators of long distance communication in vivo.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

Extracellular Vesicles Tune the Immune System in Renal Disease: A Focus on Systemic Lupus Erythematosus,Antiphospholipid Syndrome,Thrombotic Microangiopathy and ANCA-Vasculitis

Extracellular vesicles (EV) are microparticles released in biological fluids by different cell types, both in physiological and pathological conditions. Owing to their ability to carry and transfer biomolecules, EV are mediators of cell-to-cell communication and are involved in the pathogenesis of several diseases. The ability of EV to modulate the immune system, the coagulation cascade, the angiogenetic process, and to drive endothelial dysfunction plays a crucial role in the pathophysiology of both autoimmune and renal diseases. Recent studies have demonstrated the involvement of EV in the control of renal homeostasis by acting as intercellular signaling molecules, mediators of inflammation and tissue regeneration. Moreover, circulating EV and urinary EV secreted by renal cells have been investigated as potential early biomarkers of renal injury. In the present review, we discuss the recent findings on the involvement of EV in autoimmunity and in renal intercellular communication. We focused on EV-mediated interaction between the immune system and the kidney in autoimmune diseases displaying common renal damage, such as antiphospholipid syndrome, systemic lupus erythematosus, thrombotic microangiopathy, and vasculitis. Although further studies are needed to extend our knowledge on EV in renal pathology, a deeper investigation of the impact of EV in kidney autoimmune diseases may also provide insight into renal biological processes. Furthermore, EV may represent promising biomarkers of renal diseases with potential future applications as diagnostic and therapeutic tools.     

Modern Advances in CARs Therapy and Creating a New Approach to Future Treatment

Genetically engineered T and NK cells expressing a chimeric antigen receptor (CAR) are promising cytotoxic cells for the treatment of hematological malignancies and solid tumors. Despite the successful therapies using CAR-T cells, they have some disadvantages, such as cytokine release syndrome (CRS), neurotoxicity, or graft-versus-host-disease (GVHD). CAR-NK cells have lack or minimal cytokine release syndrome and neurotoxicity, but also multiple mechanisms of cytotoxic activity. NK cells are suitable for developing an “off the shelf” therapeutic product that causes little or no graft versus host disease (GvHD), but they are more sensitive to apoptosis and have low levels of gene expression compared to CAR-T cells. To avoid these adverse effects, further developments need to be considered to enhance the effectiveness of adoptive cellular immunotherapy. A promising approach to enhance the effectiveness of adoptive cellular immunotherapy is overcoming terminal differentiation or senescence and exhaustion of T cells. In this case, EVs derived from immune cells in combination therapy with drugs may be considered in the treatment of cancer patients, especially effector T and NK cells-derived exosomes with the cytotoxic activity of their original cells.     

Characteristics of Exosomes and the Vascular Landscape Regulate Exosome Sequestration by Peripheral Tissues and Brain

Exosomes mediate intercellular communication, shuttling messages between cells and tissues. We explored whether exosome tissue sequestration is determined by the exosomes or the tissues using ten radiolabeled exosomes from human or murine, cancerous or noncancerous cell lines. We measured sequestration of these exosomes by the liver, kidney, spleen, and lung after intravenous injection into male CD-1 mice. Except for kidney sequestration of three exosomes, all exosomes were incorporated by all tissues, but sequestration levels varied greatly among exosomes and tissues. Species of origin (mouse vs. human) or source (cancerous vs. noncancerous cells) did not influence tissue sequestration. Sequestration of J774A.1 exosomes by liver involved the mannose-6 phosphate (M6P) receptor. Wheatgerm agglutinin (WGA) or lipopolysaccharide (LPS) treatments enhanced sequestration of exosomes by brain and lung but inhibited sequestration by liver and spleen. Response to LPS was not predictive of response to WGA. Path and heat map analyses included our published results for brain and found distinct clusters among the exosomes and the tissues. In conclusion, we found no evidence for a universal binding site controlling exosome-tissue interactions. Instead, sequestration of exosomes by tissues is differentially regulated by both exosomes and tissues and may be stimulated or inhibited by WGA and inflammation.     

Brown Adipose Tissue Sheds Extracellular Vesicles That Carry Potential Biomarkers of Metabolic and Thermogenesis Activity Which Are Affected by High Fat Diet Intervention

Brown adipose tissue (BAT) is a key target for the development of new therapies against obesity due to its role in promoting energy expenditure; BAT secretory capacity is emerging as an important contributor to systemic effects, in which BAT extracellular vesicles (EVs) (i.e., batosomes) might be protagonists. EVs have emerged as a relevant cellular communication system and carriers of disease biomarkers. Therefore, characterization of the protein cargo of batosomes might reveal their potential as biomarkers of the metabolic activity of BAT. In this study, we are the first to isolate batosomes from lean and obese Sprague–Dawley rats, and to establish reference proteome maps. An LC-SWATH/MS analysis was also performed for comparisons with EVs secreted by white adipose tissue (subcutaneous and visceral WAT), and it showed that 60% of proteins were exclusive to BAT EVs. Precisely, batosomes of lean animals contain proteins associated with mitochondria, lipid metabolism, the electron transport chain, and the beta-oxidation pathway, and their protein cargo profile is dramatically affected by high fat diet (HFD) intervention. Thus, in obesity, batosomes are enriched with proteins involved in signal transduction, cell communication, the immune response, inflammation, thermogenesis, and potential obesity biomarkers including UCP1, Glut1, MIF, and ceruloplasmin. In conclusion, the protein cargo of BAT EVs is affected by the metabolic status and contains potential biomarkers of thermogenesis activity.     

Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.     

Brownian Motion Influence on AFM Exosomes’ Size Measurements

Extracellular vesicles are evaluated by nanoparticle tracking analysis (NTA), providing information on their hydrodynamic diameters, and by atomic force microscopy (AFM) to calculate their geometric diameters. The aim of this study is to explore the influence of Brownian movements in a sample drop and preparation time on imaging-based measurements and to determine the relationship between the geometric and hydrodynamic sizes of the extracellular vesicles measured by the AFM and the NTA, respectively. Exosomes derived from the human prostate cancer cell line PC3 were evaluated by NTA and AFM, and those results were compared with Monte Carlo simulations. The mean size, evaluated by AFM shortly after application on the mica substrate, is less than its real value. It obtains the correct value faster for a thinner sample drop. Fitting the log-normal distribution to the geometric and hydrodynamic diameters leads to the conclusion that the latter could arise from the former by linear scaling by a factor that could be used to characterize the analyzed extracellular vesicles. The size of the vesicles attached to the mica substrate depends on time. The effect of Brownian motion and stretch of the lipid bilayer should be considered in the context of exosome AFM studies.