National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2018 Annual Report of the Division of Intramural Research

Extracellular Vesicles in Pathogenesis of Human Tissue

Leonid Chernomordik
  • Leonid Margolis, PhD, Head, Section on Intercellular Interactions
  • Christophe Vanpouille, PhD, Staff Scientist
  • Anush Arakelyan, PhD, Staff Scientist
  • Rogers Nahui Palomino, BA, Visiting Fellow
  • Wendy Fitzgerald, BS, Technician
  • Vincenzo Mercurio, MS, Predoctoral Visiting Fellow

Our general goal is to understand the mechanisms of pathogenesis and transmission of human pathogens, in particular of the human immunodeficiency virus (HIV). Over the last several years, it has become clear that infected cells release not only viral particles but also extracellular vesicles (EVs) of viral size that play a role in viral infection. Moreover, the release of EVs is a part of normal cell physiology, and EVs play an important role in cell-cell communication. During the past year, our efforts focused on establishing the role of EVs in health and disease, in particular in viral infection. It is important to study the role of EVs in the context of human tissues, in which the critical events of many human diseases occur. Our Section was a pioneer in establishing new experimental tissue system in which pathogenesis can be studied in in vivo–like systems under controlled laboratory conditions. Also, such systems are now used as a platform in preclinical testing of antivirals.

This year, we continued to develop such systems, in particular, a system of human placenta ex vivo to study its physiology in health and disease. Similarly, we developed a system of ex vivo atherosclerotic plaques to study atherogenesis under controlled laboratory conditions. In such systems, tissue cyto-architecture as well as native cell-cell communications are largely preserved. One of the main means of such communication are cytokines that are release by various cells. We investigated the role of EVs as carriers of cytokines and their role in viral infection in different systems of ex vivo tissues and in several body fluids. In particular, we found that, although cytokines are generally considered to function as classical soluble molecules mediating cell-cell communications in multicellular organisms, bioactive cytokines are also released in association with EVs, in particular encapsulated in EVs. The finding is important for our understanding of the mechanisms of cell-cell communication in tissues and may lead to a reconsideration of the analysis of cytokines in immuno-activated tissues, given that, by standard protocols of cytokine measurements, cytokines are not detectable within EVs.

We continued to investigate the role of viral infections on EVs. In work published in 2017, we showed that, in addition to infectious viral particles, HIV–infected cells release EVs that carry viral molecules, in particular membrane molecules. We hypothesized that the release of EVs carrying viral proteins is a general phenomenon not restricted to HIV. This year, in support of this hypothesis, we found that cells infected with human cytomegalovirus (hCMV) also release EVs that carry hCMV membrane proteins that play an important role in viral infection.

Extracellular vesicles in hCMV infection

Human cytomegalovirus (hCMV) is an important pathogen and is implicated in immune stimulation in the course of HIV infection, even after productive HIV infection is fully suppressed. We investigated whether infected cells release EVs that carry hCMV proteins and may therefore play a role in viral pathogenesis, including host immune response. We studied EVs isolated from the cell-free supernatant of human lung fibroblasts (MRC-5 cells) infected with a recombinant hCMV labeled with an enhanced green fluorescent protein [EGFP] or of primary dermal fibroblast cells infected with the AD169 strain of hCMV, using an iodixanol step-gradient (a radiocontrast, non-osmotic density gradient medium). We determined the purity of this fraction by measuring hCMV DNA by qPCR and the size and distribution of EVs using Nanosight and transmission electron microscopy (TEM). Also, to identify contaminating hCMV virions, we stained all lipid-containing particles in EGFP-hCMV viral preparations with the fluorescent dye DiI, which stains everything that has a lipid membrane. In our preparation, this was either viral particles (already labeled with EGFP) or vesicles. Thus, viruses become labeled with both dyes whereas vesicles with only one. This analysis of individual particles allowed us to distinguish between hCMV virions and EVs. Using specific fluorescent antibodies, we also analyzed the expression of gB and gH, two abundant envelope glycoproteins of hCMV, on EVs. We found that 15% of EVs were positive for gB and 5% were positive for gH, a proportion consistent with the relative representation of these glycoproteins in the envelope of the mature cell-free virus. A smaller fraction (4%) was positive for both viral proteins. We confirmed this conclusion with a flow technique, originally developed in our laboratory, that allows us to analyze individual EVs based on their capture by 15-nm magnetic nanoparticles (MNPs) coupled to monoclonal antibodies. Specifically, we captured DiI–stained EVs, collected from infected or control uninfected MRC-5 cells, and purified them on an iodixanol density gradient, with MNPs coupled to specific anti-gB antibodies and labeled with the fluorescent probe Zenon AF488. To avoid aggregation, we used MNPs in large excess over the number of virions or EVs. We isolated the EV–MNP complexes on magnetic columns and eluted, and visualized them with a flow cytometer. Most of the GFP–negative events were double-positive for DiI and anti-gB AF488 antibodies, thus representing captured gB–positive EVs. Thresholding on AF488 fluorescence revealed a similar number of EVs. In control experiments using uninfected MRC-5 cells, we found no EVs carrying gB. Thus, similar to EVs released by HIV–infected cells, EVs released by hCMV–infected cells carry viral surface proteins. Such EVs may contribute to various physiological effects in which viruses have been implicated, given that these EVs and hCMV should target the same cells, i.e., cells expressing hCMV receptors.

Extracellular vesicles as cytokine carriers

We undertook a first comprehensive study of cytokine association with EVs in an attempt to answer the following questions: (1) how general the phenomenon of cytokine association with EVs is; (2) whether only particular cytokines are associated with EVs; (3) whether a cytokine released in association with EVs in one system can be released as a free (soluble) molecule in another; (4) whether the association of cytokines with EVs is a regulated process that can be modulated; (5) whether cytokines are encapsulated in EVs rather than merely attached to them; and (6) whether EV–encapsulated cytokines can be delivered to sensitive cells and trigger a physiological response.

To answer these questions, we systematically analyzed the association between 33 cytokines and EVs in eight in vitro, ex vivo, and in vivo biological systems (cultured T cells, cultured monocytes, explants of tonsillar, cervical, placental villous, and amnion tissues, amniotic fluid, and blood plasma of healthy volunteers). We demonstrated that the association of a given cytokine with EVs is not necessarily linked to the property of the cytokine but rather to the regulated property of a system. In the eight different systems, we found that a given cytokine can be released predominantly in a soluble form in one system, while in another it can be predominantly EV–associated. For example, the chemo-attracting cytokine MIG (monokine induced by gamma interferon) in the plasma of healthy donors and in amniotic fluid is present almost exclusively in a soluble form, while both monocytes and T cells release this cytokine almost exclusively in association with EVs. This is true also for several other cytokines. In general, placental villous explants secreted cytokines preferentially in soluble forms, including eight cytokines that were found over 90% in the free form. In contrast, T cells and monocytes released some of the same cytokines predominantly associated with EVs. The difference between placental villous explants and immune cells may be related to the high level of expression of cytokines by these tissue explants, while T cell and monocyte suspensions that express much lower levels of cytokines may need to concentrate them in EVs rather than dissolving them in solution.

Among EV–associated cytokines, IL-2, IL-4, IL-10, IL-12, IL-15, IL-16, IL-18, IL-21, IL-22, IL-33, Eotaxin, IP-10, ITAC, M-CSF, MIG, MIP-3, TGF-beta, and TNF-alpha were preferentially encapsulated in EVs across all systems. In some instances, all EV–associated cytokine was inside EVs (for example, IL-10 in cervix, T cell and monocyte cultures, and IFN-gamma in placental villous and amnion cultures, as well as in amniotic fluid). Thus, different biological systems differentially distribute the released cytokines between free and EV–associated forms. The pattern of the cytokine released is not a fixed property of the system but rather can be modulated: both tonsillar explants and cultured monocytes altered the relative fractions of free and EV–associated forms of cytokines upon activation. Moreover, the two stimuli used here to activate monocytes dramatically changed the pattern of cytokine association with EVs. Thus, the pattern of cytokine packaging in EVs strongly depends on the nature of the activator.

The nature of cytokine association with EVs also depends on the system and its activation state. For example, placenta release EV in which IL-22 is predominantly encapsulated while IL-13 is predominantly exposed on the outer EV surface for EVs released by the same tissue. The entire distribution of cytokines between inner and outer EV compartments is not constant but changes upon activation of the systems by different stimuli. Even though a significant fraction of cytokines are encapsulated in EVs and are thus not detected by standard target cell-free cytokine assays, these cytokines may play important physiological roles. Therefore, the interpretations of the roles of these cytokines in health and disease based on these standard assays should be now reconsidered. The system of EV–encapsulated cytokines we described may represent an important system of cell-cell communication in health and disease and may serve as a new therapeutic target.

Development of new ex vivo tissue systems to study pathogenesis

To study normal and pathologic cell interactions in tissues, as well as various tissue infections, it is important to develop adequate models that allow one to study these processes under controlled laboratory conditions. Earlier, we developed a model of lymphoid tissue ex vivo that is used to study HIV pathogenesis. We took advantage of this unique system of tissue culture to test dual-targeted compounds that inhibit HIV and TB infection. Also, we developed two new systems to study important aspect of human pathology. (1) To study the mechanisms of placenta function and the role of EVs in pregnancy, we developed an ex vivo system that retains placental cyto-architecture and its main metabolic aspects, in particular the release of EVs and soluble factors. (2) To study mechanisms of atherosclerosis, we developed an ex vivo system of atherosclerotic plaques.

(1) We used the ex vivo system of human placenta to investigate the pattern of secretion of cytokines, growth factors, and EVs by placental villous and amnion tissues. We cultured placental villous and amnion explants for two weeks at the air/liquid interface and analyzed their morphology and the released cytokines and EVs. Placental explants of both placental villous tissue and amnion were viable for at least 14 days. Both types of explants continue to secrete cytokines and growth factors over 14 days of culture, providing further evidence of tissue viability and functioning. We found that syncytiotrophoblast-specific EVs can be captured from placental villous culture supernatants by MNPs coupled to antibodies against specific placental antigens. These EVs carried the membrane proteins CD51, CD63, CD105, CD200, CD274, and syncytin-1. EVs produced by amnion and captured with anti-CD90 MNPs expressed CD29, CD44, CD105, CD140b, CD324, and CD326, which are involved in cell-cell and cell-matrix interactions, as well as cell adhesion, and migration. We investigated the expression of cytokines that were associated with EVs generated by placenta amnion and villous parts. The complex differential distribution of cytokines between EVs of different origin and phenotype suggests a fine regulation of their biogenesis and different biological functions. In general, a system of ex vivo placental villous and amnion tissues can be used as an adequate model to study placental metabolic activity in normal and complicated pregnancies.

Additional Funding

  • Office of AIDS Research (OAR), NIH, Intramural Award

Publications

  1. AFitzgerald W, Freeman ML, Lederman MM, Vasilieva E, Romero R, Margolis L. A system of cytokines encapsulated in extracellular vesicles. Sci Rep 2018;8:8973.
  2. Fitzgerald W, Gomez-Lopez N, Erez O, Romero R, Margolis L. Extracellular vesicles generated by placental tissues ex vivo: a transport system for immune mediators and growth factors. Am J Reprod Immunol 2018;80:e12860.
  3. Zicari S, Arakelyan A, Palomino R, Fitzgerald W, Vanpouille C, Lebedeva A, Schmitt A, Bomsel M, Britt W, Margolis L. Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins. Virology 2018;524:97-105.
  4. Arakelyan A, Fitzgerald W, Zicari S, Vanpouille C, Margolis L. Extracellular vesicles carry HIV Env and facilitate Hiv infection of human lymphoid tissue. Sci Rep 2017;7:1695.
  5. Ñahui Palomino RA, Zicari S, Vanpouille C, Vitali B, Margolis L. Vaginal Lactobacillus inhibits HIV-1 replication in human tissues ex vivo. Front Microbiol 2017;8:906.

Collaborators

  • Morgan Bomsel, PhD, Institut Cochin, Paris, France
  • William Britt, MD, University of Alabama School of Medicine, Birmingham, AL
  • Leonid Chernomordik, PhD, Section on Membrane Biology, NICHD, Bethesda, MD
  • Sara Gianella Weibel, MD, University of California San Diego, La Jolla, CA
  • Sergey Kochetkov, PhD, Engelhard Institute of Molecular Biology, Moscow, Russia
  • Michael Lederman, MD, Case Western University, Cleveland, OH
  • David D. Roberts, PhD, Laboratory of Pathology, Center for Cancer Research, NCI, Bethesda, MD
  • Roberto Romero-Galue, MD, DMedSci, Perinatology Research Branch, NICHD, Detroit, MI
  • Alexandr Shpektor, MD, Moscow Medical University, Moscow, Russia
  • Elena Vasilieva, MD, Moscow Medical University, Moscow, Russia
  • Beatrice Vitali, PhD, Università di Bologna, Bologna, Italy

Contact

For more information, email margolis@helix.nih.gov or visit http://irp.nih.gov/pi/leonid-margolis.

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