Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion.

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.

Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation.

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

U6/miR-211 expression ratio as a purity parameter for HEK293 cell-derived exosomes.

Small extracellular vesicles (sEVs) including exosomes are produced by all cell types and can be isolated from biological fluids and cell culture supernatants. The separation of exosomes with high purity from protein-rich media remains challenging. Besides contaminating proteins, small microvesicles (MVs) and apoptotic bodies are usually co-isolated with exosomes. The optimization of exosome separation and purification depends on reliable methods for the determination of the purity of the preparation, but no standard measurement has been defined so far. We tried to advance purity assessment. sEVs were isolated from HEK293 cell culture supernatants by various combinations of centrifugation, precipitation and size exclusion chromatography. sEVs with a diameter within the size range of 30-150 nm, typical for exosomes, were obtained with all tested isolation methods as shown by electron microscopy. To estimate the levels of protein contamination, flow cytometric analysis of the obtained vesicles was used. Based on the controlled preferential loading and enrichment of miR-211 into exosomes, a novel approach for the estimation of the fraction of HEK293 derived exosomes as opposed to MVs and apoptotic bodies in sEV mixtures was developed. This novel approach represents a simple qRT-PCR-based approach to improve the precise characterization of sEV isolates that is necessary for the usage of exosomes as carriers for therapeutic nucleic acids. Compared to the precipitation and size exclusion chromatography, the differential ultracentrifugation turned out to give sEVs with fairly intact shape and the highest purity according to the novel qRT-PCR-based approach, as well as to other established methods for purification assessment.

Intracellular HINT1-Assisted Hydrolysis of Nucleoside 5'-O-Selenophosphate Leads to the Release of Hydrogen Selenide That Exhibits Toxic Effects in Human Cervical Cancer Cells.

In this study, we present a new selenium derivative, 2'-deoxyguanosine-5'-O-selenophosphate (dGMPSe), synthesized by the oxathiaphospholane method and adapted here for the synthesis of nucleoside selenophosphates. Using biochemical assays (HPLC- and fluorescence-based), we investigated the enzymatic activity of HINT1 towards dGMPSe in comparison with the corresponding thiophosphate nucleoside, i.e., dGMPS. Both substrates showed similar kcat and a small difference in Km, and during the reactions the release of reducing agents such as H2Se and H2S were expected and detected. MTT viability assay and microscopic analysis showed that dGMPSe was toxic to HeLa cancer cells, and this cytotoxicity was due to the release of H2Se. The release of H2Se or H2S in the living cells after administration of dGMPSe and/or dGMPS, both without carrier and by electroporation, was observed using a fluorescence assay, as previously for NMPS. In conclusion, our comparative experiments with dGMPSe and dGMPS indicate that the HINT1 enzyme is capable of converting (d)NMPSe to (d)NMP and H2Se, both in vitro and intracellularly. Since the anticancer activity of various selenium compounds depends on the formation of hydrogen selenide, the actual inducer of cell death, we propose that selenium-containing nucleotides represent another option as novel compounds with anticancer therapeutic potential.

EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles.

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.

Exosomes as Messengers Between Mother and Fetus in Pregnancy.

The ability of exosomes to transport different molecular cargoes and their ability to influence various physiological factors is already well known. An exciting area of research explores the functions of exosomes in healthy and pathological pregnancies. Placenta-derived exosomes were identified in the maternal circulation during pregnancy and their contribution in the crosstalk between mother and fetus are now starting to become defined. In this review, we will try to summarize actual knowledge about this topic and to answer the question of how important exosomes are for a healthy pregnancy.

Methods for the Determination of the Purity of Exosomes.

BACKGROUND: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance. METHODS: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database. RESULTS: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols. CONCLUSION: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.

Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance.

BACKGROUND: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. METHODS: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. RESULTS: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-ß transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. CONCLUSION: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

Analysis of the miRNA Profiles of Melanoma Exosomes Derived Under Normoxic and Hypoxic Culture Conditions.

BACKGROUND/AIM: MicroRNAs (miRNAs) transported in melanoma-derived exosomes function as intercellular messengers supporting tumor survival and progression. Hypoxia increases melanoma phenotypic plasticity, drug resistance, and metastasis. MATERIALS AND METHODS: We determined the miRNA profiles in exosomes derived from melanoma cells grown under hypoxic and normoxic conditions by microarray analyses and reverse transcription-polymerase chain reaction (RT-PCR) in order to analyze the potential influence of vesicle-transported miRNAs on cancer-related pathways and transcriptional programs. RESULTS: Despite phenotypical differences of the four cell lines used, their exosomes shared the majority of miRNAs. The levels of three miRNAs were higher in normoxic exosomes, whereas 15 miRNAs were significantly more abundant under hypoxic conditions. Pathway analysis pointed at several cellular processes contributing to proliferation, drug resistance, and modification of the tumor microenvironment, including immunosuppression. CONCLUSION: The miRNA-expression profiles of exosomes from patient-derived melanoma cells are modified by oxygen concentration and reflect the phenotypic changes of melanoma cells under different growth conditions.

Functions of Cancer-Derived Extracellular Vesicles in Immunosuppression.

Extracellular vesicles, including exosomes, constitute an important element of intercellular communication by carrying a variety of molecules from producer to target cells. The transport of mRNA and miRNA can directly modulate gene expression in the target cells. The miRNA content in exosomes is characteristic for the cell from which the vesicles were derived enabling the usage of exosomes as biomarkers for the diagnosis various diseases, including cancer. Cancer-derived exosomes support the survival and progression of tumors in many ways and also contribute to the neutralization of the anti-cancer immune response. Exosomes participate in all known mechanisms by which cancer evades the immune system. They influence the differentiation and activation of immune suppressor cells, they modulate antigen presentation, and are able to induce T-cell apoptosis. Although cancer-derived exosomes mainly suppress the immune system and facilitate tumor progression, they are also important sources of tumor antigens with potential clinical application in stimulating immune responses. This review summarizes how exosomes assist cancer to escape immune recognition and to acquire control over the immune system.