Subpopulations of extracellular vesicles and their therapeutic potential

Lä,sser, Cecilia,Jang, Su Chul,Lö,tvall, Jan Elsevier 2018 Molecular aspects of medicine Vol.60 No.-

<P><B>Abstract</B></P> <P>Extracellular vesicles (EVs), such as exosomes and microvesicles, have over the last 10–15 years been recognized to convey key messages in the molecular communication between cells. Indeed, EVs have the capacity to shuttle proteins, lipids, and nucleotides such as RNA between cells, leading to an array of functional changes in the recipient cells. Importantly, the EV secretome changes significantly in diseased cells and under conditions of cellular stress. More recently, it has become evident that the EV secretome is exceptionally diverse, with many different types of EVs being released by a single cell type, and these EVs can be described in terms of differences in density, molecular cargos, and morphology. This review will discuss the diversity of EVs, will introduce some suggestions for how to categorize them, and will propose how EVs and their subpopulations might be used for very different therapeutic purposes.</P>

Pulmonary inflammation induced by bacteria-free outer membrane vesicles from Pseudomonas aeruginosa.

Park, Kyong-Su,Lee, Jaewook,Jang, Su Chul,Kim, Sae Rom,Jang, Myoung Ho,L?tvall, Jan,Kim, Yoon-Keun,Gho, Yong Song The Association 2013 American journal of respiratory cell and molecular Vol.49 No.4

<P>Pseudomonas aeruginosa is often involved in lung diseases such as cystic fibrosis. These bacteria can release outer membrane vesicles (OMVs), which are bilayered proteolipids with diameters of approximately 20 to 250 nm. In vitro, these OMVs activate macrophages and airway epithelial cells. The aim of this study was to determine whether OMVs from P. aeruginosa can induce pulmonary inflammation in vivo and to elucidate the mechanisms involved. Bacteria-free OMVs were isolated from P. aeruginosa cultures. Wild-type, Toll-like receptor (TLR)2 and TLR4 knockout mice were exposed to OMVs by the airway, and inflammation in the lung was assessed using differential counts, histology, and quantification of chemokines and cytokines. The involvement of the TLR2 and TLR4 pathways was studied in human cells using transfection. OMVs given to the mouse lung caused dose- and time-dependent pulmonary cellular inflammation. Furthermore, OMVs increased concentrations of several chemokines and cytokines in the mouse lungs and mouse alveolar macrophages. The inflammatory responses to OMVs were comparable to those of live bacteria and were only partly regulated by the TLR2 and TLR4 pathways, according to studies in knockout mice. This study shows that OMVs from P. aeruginosa cause pulmonary inflammation without live bacteria in vivo. This effect is only partly controlled by TLR2 and TLR4. The role of OMVs in clinical disease warrants further studies because targeting of OMVs in addition to live bacteria may add clinical benefit compared with treating with antibiotics alone.</P>

Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

Karimi, Nasibeh,Cvjetkovic, Aleksander,Jang, Su Chul,Crescitelli, Rossella,Hosseinpour Feizi, Mohammad Ali,Nieuwland, Rienk,Lö,tvall, Jan,Lä,sser, Cecilia Springer International Publishing 2018 Cellular and Molecular Life Sciences Vol.75 No.15

<P>The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1007/s00018-018-2773-4) contains supplementary material, which is available to authorized users.</P>

Bacterial Protoplast-Derived Nanovesicles as Vaccine Delivery System against Bacterial Infection

Kim, Oh Youn,Choi, Seng Jin,Jang, Su Chul,Park, Kyong-Su,Kim, Sae Rom,Choi, Jun Pyo,Lim, Ji Hwan,Lee, Seung-Woo,Park, Jaesung,Di Vizio, Dolores,Lö,tvall, Jan,Kim, Yoon-Keun,Gho, Yong Song American Chemical Society 2015 NANO LETTERS Vol.15 No.1

<P>The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immune-stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles in Gram-negative bacteria, have gained attention for the next generation vaccine. However, the more invasive and effective the vaccine is in delivery, the more risk it holds for severe immune toxicity. Here, in optimizing the current vaccine delivery system, we designed bacterial protoplast-derived nanovesicles (PDNVs), depleted of toxic outer membrane components to generate a universal adjuvant-free vaccine delivery system. These PDNVs exhibited significantly higher productivity and safety than the currently used vaccine delivery vehicles and induced strong antigen-specific humoral and cellular immune responses. Moreover, immunization with PDNVs loaded with bacterial antigens conferred effective protection against bacterial sepsis in mice. These nonliving nanovesicles derived from bacterial protoplast open up a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines to be used to prevent infectious diseases.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/nalefd/2015/nalefd.2015.15.issue-1/nl503508h/production/images/medium/nl-2014-03508h_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nl503508h'>ACS Electronic Supporting Info</A></P>

Immunization with <i>Escherichia coli</i> Outer Membrane Vesicles Protects Bacteria<i>-</i>Induced Lethality via Th1 and Th17 Cell Responses

Kim, Oh Youn,Hong, Bok Sil,Park, Kyong-Su,Yoon, Yae Jin,Choi, Seng Jin,Lee, Won Hee,Roh, Tae-Young,Lö,tvall, Jan,Kim, Yoon-Keun,Gho, Yong Song The American Association of Immunologists, Inc. 2013 JOURNAL OF IMMUNOLOGY Vol.190 No.8

<P>Outer membrane vesicles (OMVs), secreted from Gram-negative bacteria, are spherical nanometer-sized proteolipids enriched with outer membrane proteins. OMVs, also known as extracellular vesicles, have gained interests for use as nonliving complex vaccines and have been examined for immune-stimulating effects. However, the detailed mechanism on how OMVs elicit the vaccination effect has not been studied extensively. In this study, we investigated the immunological mechanism governing the protective immune response of OMV vaccines. Immunization with <I>Escherichia coli</I>–derived OMVs prevented bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome. As verified by adoptive transfer and gene-knockout studies, the protective effect of OMV immunization was found to be primarily by the stimulation of T cell immunity rather than B cell immunity, especially by the OMV-Ag–specific production of IFN-γ and IL-17 from T cells. By testing the bacteria-killing ability of macrophages, we also demonstrated that IFN-γ and IL-17 production is the main factor promoting bacterial clearances. Our findings reveal that <I>E. coli</I>–derived OMV immunization effectively protects bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome primarily via Th1 and Th17 cell responses. This study therefore provides a new perspective on the immunological detail regarding OMV vaccination.</P>

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing

Lä,sser, Cecilia,Shelke, Ganesh Vilas,Yeri, Ashish,Kim, Dae-Kyum,Crescitelli, Rossella,Raimondo, Stefania,Sjö,strand, Margareta,Gho, Yong Song,Van Keuren Jensen, Kendall,Lö,tvall, Jan TaylorFrancis 2017 RNA BIOLOGY Vol.14 No.1

<P><B>ABSTRACT</B></P><P>Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion.</P><P>We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.</P>

Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delivery of Chemotherapeutics to Malignant Tumors

Jang, Su Chul,Kim, Oh Youn,Yoon, Chang Min,Choi, Dong-Sic,Roh, Tae-Young,Park, Jaesung,Nilsson, Jonas,Lö,tvall, Jan,Kim, Yoon-Keun,Gho, Yong Song American Chemical Society 2013 ACS NANO Vol.7 No.9

<P>Exosomes, the endogenous nanocarriers that can deliver biological information between cells, were recently introduced as new kind of drug delivery system. However, mammalian cells release relatively low quantities of exosomes, and purification of exosomes is difficult. Here, we developed bioinspired exosome-mimetic nanovesicles that deliver chemotherapeutics to the tumor tissue after systemic administration. The chemotherapeutics-loaded nanovesicles were produced by the breakdown of monocytes or macrophages using a serial extrusion through filters with diminishing pore sizes (10, 5, and 1 μm). These cell-derived nanovesicles have similar characteristics with the exosomes but have 100-fold higher production yield. Furthermore, the nanovesicles have natural targeting ability of cells by maintaining the topology of plasma membrane proteins. <I>In vitro</I>, chemotherapeutic drug-loaded nanovesicles induced TNF-α-stimulated endothelial cell death in a dose-dependent manner. <I>In vivo</I>, experiments in mice showed that the chemotherapeutic drug-loaded nanovesicles traffic to tumor tissue and reduce tumor growth without the adverse effects observed with equipotent free drug. Furthermore, compared with doxorubicin-loaded exosomes, doxorubicin-loaded nanovesicles showed similar <I>in vivo</I> antitumor activity. However, doxorubicin-loaded liposomes that did not carry targeting proteins were inefficient in reducing tumor growth. Importantly, removal of the plasma membrane proteins by trypsinization eliminated the therapeutic effects of the nanovesicles both <I>in vitro</I> and <I>in vivo</I>. Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome-mimetics to effectively deliver chemotherapeutics to treat malignant tumors.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancac3/2013/ancac3.2013.7.issue-9/nn402232g/production/images/medium/nn-2013-02232g_0010.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nn402232g'>ACS Electronic Supporting Info</A></P>