1. Extracellular vesicles in hCMV infection. Human cytomegalovirus (hCMV) is an important pathogen that 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 therefore may play a role in viral pathogenesis including host immune response. We studied EVs isolated from the cell-free supernatant of UL32-EGFP-HCMV-infected human lung fibroblast or AD169 HCMV-infected primary dermal fibroblast cells using an iodixanol step-gradient. We determined the purity of this fraction by measuring HCMV DNA by qPCR and the size and distribution using Nanosight and transmission electron microscopy. To identify contaminating hCMV virions, we stained all lipid containing particles in a UL32-EGFP-hCMV viral preparations with a fluorescent dye, DiI. Therefore, by thresholding on DiI, any event positive for DiI and GFP represented a virion and any DiI-positive / GFP-negative event represented a vesicle. This analysis of individual particles allowed us to distinguish HCMV virions from EVs. Finally, using specific fluorescent antibodies, we 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. This conclusion was confirmed with a flow technique originally developed in our laboratory, which allows us to analyze antigenic composition of individual particles. 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 since these EVs and hCMV should target the same cells., i.e. cells expressing hCMV receptors. 2. Extracellular vesicles as cytokine carriers We undertook a comprehensive study of cytokine association with EVs in an attempt to answer the following questions: (i) how general is the phenomenon of cytokine association with EVs? (ii) are only particular cytokines associated with EVs? (iii) can a cytokine released in association with EVs in one system get released as a free (soluble) molecule in another? (iv) is the association of cytokines with EVs a regulated process that can be modulated? (v) are cytokines encapsulated in EVs rather than merely attached to them? (vi) can EV-encapsulated cytokines be delivered to sensitive cells and trigger a physiological response? 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 tonsils, cervical, placental villous, and amnion tissues, amniotic fluid, and blood plasma of healthy volunteers). We demonstrated that being associated with EVs is not necessarily the property of a cytokine but rather is the regulated property of a system. In the eight different systems we studied, a given cytokine in one system can be released predominantly in a soluble form, while in another it can be predominantly EV-associated. Indeed, placental villous explants preferentially elaborated more cytokines in soluble form, 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-b, and TNF- 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- 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 changed the pattern of cytokine association with EVs in dramatically different patterns. In spite of a significant fraction of cytokines being encapsulated in EVs, these cytokines are not detected by standard target cell-free cytokine assays. 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 may represent an important system of cell-cell communication in health and disease and may serve as a new therapeutic target. 3. Developing 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 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: (i) To study the mechanisms of placenta function and the role of EVs in pregnancy, we developed an ex vivo system that retains placental cytoarchitecture and the main metabolic aspects, in particular the release of EVs and soluble factors. (ii) To study mechanisms of atherosclerosis, we developed an ex vivo system of atherosclerotic plaques. (i) We developed an ex vivo system of human placenta and investigated the pattern of secretion of cytokines, growth factors and extracellular vesicles by placental villous and amnion tissues ex vivo. Placental villous and amnion explants were cultured for two weeks at the air/liquid interface and their morphology and the released cytokines and EVs were analyzed. 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 using magnetic nanoparticles (MNP) coupled to antibodies against specific placenta antigens. These EVs carried, 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 placenta metabolic activity in normal and complicated pregnancies, and to provide new insight in the functional status in various placental disorders.
| Fitzgerald, Wendy; Gomez-Lopez, Nardhy; Erez, Offer et al. (2018) Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors. Am J Reprod Immunol 80:e12860 |
| Introini, Andrea; Fitzgerald, Wendy; Vanpouille, Christophe et al. (2018) Histoculture and Infection with HIV of Functional Human Lymphoid Tissue on Gelfoam®. Methods Mol Biol 1760:187-197 |
| Kaur, Sukhbir; Elkahloun, Abdel G; Arakelyan, Anush et al. (2018) CD63, MHC class 1, and CD47 identify subsets of extracellular vesicles containing distinct populations of noncoding RNAs. Sci Rep 8:2577 |
| Fitzgerald, Wendy; Freeman, Michael L; Lederman, Michael M et al. (2018) A System of Cytokines Encapsulated in ExtraCellular Vesicles. Sci Rep 8:8973 |
| Zicari, Sonia; Arakelyan, Anush; Palomino, Rogers Alberto Ñahui et al. (2018) Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins. Virology 524:97-105 |
| Christensen-Quick, Aaron; Vanpouille, Christophe; Lisco, Andrea et al. (2017) Cytomegalovirus and HIV Persistence: Pouring Gas on the Fire. AIDS Res Hum Retroviruses 33:S23-S30 |
| Derudas, Marco; Vanpouille, Christophe; Carta, Davide et al. (2017) Virtual Screening of Acyclovir Derivatives as Potential Antiviral Agents: Design, Synthesis, and Biological Evaluation of New Acyclic Nucleoside ProTides. J Med Chem 60:7876-7896 |
| Alexandrova, Liudmila; Zicari, Sonia; Matyugina, Elena et al. (2017) Dual-targeted anti-TB/anti-HIV heterodimers. Antiviral Res 145:175-183 |
| Vagida, Murad; Arakelyan, Anush; Lebedeva, Anna et al. (2017) Flow analysis of individual blood extracellular vesicles in acute coronary syndrome. Platelets 28:165-173 |
| Tarca, Adi L; Fitzgerald, Wendy; Chaemsaithong, Piya et al. (2017) The cytokine network in women with an asymptomatic short cervix and the risk of preterm delivery. Am J Reprod Immunol 78: |
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