Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926

Autoři: Myriam Lucia Velandia-Romero aff001;  María Angélica Calderón-Peláez aff001;  Arturo Balbás-Tepedino aff001;  Ricaurte Alejandro Márquez-Ortiz aff002;  L. Johana Madroñero aff002;  Alfonso Barreto Prieto aff003;  Jaime E. Castellanos aff001
Působiště autorů: Grupo de Virología, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, Colombia aff001;  Laboratorio Genética Molecular Bacteriana, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, Colombia aff002;  Grupo de Inmunobiología y Biología Celular, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227030


Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.

Klíčová slova:

Apoptosis – Endothelial cells – Exosomes – Immunoprecipitation – Macrophages – Permeability – Vesicles – RNA transport


1. Yoon Y, Kim O, Gho Y. Extracellular vesicles as emerging intercellular communicasomes. BMB Rep. 2014; 47: 531–39. doi: 10.5483/BMBRep.2014.47.10.164

2. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, macrovesicles and friends. J Cell Biol. 2013; 200: 373–83. doi: 10.1083/jcb.201211138 23420871

3. Revenfeld A, Baek R, Nielsen MH, Stensballe A, Varming K, Jorgersen M. Diagnostic and prognostic potential of extracellular vesicles in peripheral blood. Clin Ther. 2014; 36: 830–46. doi: 10.1016/j.clinthera.2014.05.008 24952934

4. Tokarev A, Alfonso A, Segev N. Overview of Intracellular Compartments and Trafficking Pathways. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000–2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7286/

5. van der Pol E, Böing A, Harrinson P, Sturk A, Nieuwland R. Classification, functions and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012; 64: 676–05. doi: 10.1124/pr.112.005983 22722893

6. Colombo M, Raposo G, Théry C. Biogenesis, secretion and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014; 30: 255–89. doi: 10.1146/annurev-cellbio-101512-122326 25288114

7. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006; Chapter 3, Unit 3.22: 3.22.1–3.22.29. doi: 10.1002/0471143030.cb0322s30

8. Vlassov A, Magdaleno S, Setterquist R; Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutically potentials. Biochim Biophys Acta. 2012; 1820: 940–48. doi: 10.1016/j.bbagen.2012.03.017 22503788

9. Jiang L, Paone S, Caruso S, Atkin-Smith G, Phan T, Hulett M, et al. Determining the contents and cell origins of apoptotic bodies by flow cytometry. Sci Rep. 2017; 7: 14444. doi: 10.1038/s41598-017-14305-z 29089562

10. Hauser P, Wang S, Didenko V. Apoptotic Bodies: selective detection in extracellular vesicles. Methods Mol Biol. 2017; 1554: 193–00. doi: 10.1007/978-1-4939-6759-9_12 28185192

11. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune response. Nat Rev Immunol. 2009; 9: 581–93. doi: 10.1038/nri2567 19498381

12. Atkin-Smith G, Paone S, Zanker D, Duan M, Phan T, Chen W, et al. Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting. Sci Rep. 2017; 7: 39846. doi: 10.1038/srep39846 28057919

13. György B, Szabó T, Pásztói M, Pál Z, Misják P, Aradi B, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci. 2011; 68: 2667–88. doi: 10.1007/s00018-011-0689-3 21560073

14. Braciale T, Hahn Y. Immunity to viruses. Immunol Rev. 2013, 255: 5–12. doi: 10.1111/imr.12109 23947343

15. Pelchen-Matthews A, Raposo G, Marsh M. Endosomes, exosomes and trojan viruses. Trends Microbiol. 2004; 12: 310–16. doi: 10.1016/j.tim.2004.05.004 15223058

16. Barreto A, Rodríguez LS, Rojas O, Wolf M, Greenberg H, Franco M, et al. Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function. Viral Immunol. 2010; 23: 595–08. doi: 10.1089/vim.2009.0113 21142445

17. Goswami S, Banerjee A, Kumari B, Bandopadhyay B, Bhattacharya N, Basu N, et al. Differential expression and significance of circulating microRNAs in cerebrospinal fluid of acute encephalitis patients infected with Japanese Encephalitis Virus. Mol Neurobiol. 2017; 54: 1541–51. doi: 10.1007/s12035-016-9764-y 26860411

18. Flanagan J, Middeldorp J, Sculley T. Localization of the Epstein-Barr virus protein LMP1 to exosomes. J Gen Virol. 2003; 84: 1871–79. doi: 10.1099/vir.0.18944-0 12810882

19. Chahar H, Bao X, Casola A. Exosomes and their role in the life cycle and pathogenesis. Viruses. 2015; 7: 3204–25. doi: 10.3390/v7062770 26102580

20. Kapoor N, Kumar V. Emerging Role of exosomal secretory pathway in human tumor virus pathogenesis. Int J Biochem Res Rev. 2014; 4: 653–65. doi: 10.9734/IJBcRR/2014/9370

21. Malik S, Eugenin E. Mechanisms of HIV neuropathogenesis: role of cellular communication systems. Curr HIV Res. 2016; 14: 400–11. doi: 10.2174/1570162x14666160324124558 27009098

22. Khan M, Lang M, Huang M, Raymond A, Bond V, Shiramizu B, et al. Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ 1–42 secretion in SH-SY5Y neural cells. J Neurovirol. 2016; 22: 179–90. doi: 10.1007/s13365-015-0383-6 26407718

23. Lin Z, Swan K, Zhang X, Cao S, Brett Z, Drury S, et al. Secreted oral epithelial cell membrane vesicles induce Epstein-Barr Virus reactivation in latently infected B cells. J Virol. 2016; 90: 3469–79. doi: 10.1128/JVI.02830-15 26764001

24. Lai C, Saxena V, Tseng C, Jeng K, Kohara M, Lai M. Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport. Plos One. 2014; 9: e99022. doi: 10.1371/journal.pone.0099022 24905011

25. Masciopinto F, Giovani C, Campagnoli S, Galli-Stapino L, Colombatto, Brunetto M, et al. Association of hepatitis C virus envelope proteins with exosomes. Eur J Immunol. 2004; 34: 2834–42. doi: 10.1002/eji.200424887 15368299

26. Bukong T, Momen-Heravi F, Kodys K, Bala S, Szabo G. Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS Pathogens. 2014; 10: e1004424. doi: 10.1371/journal.ppat.1004424 25275643

27. Liu Z, Zhang X, Yu Q, He J. Exosome-associated hepatitis C virus in cell cultures and patient plasma. Biochem Biophys Res Commun. 2014; 455: 218–22. doi: 10.1016/j.bbrc.2014.10.146 25449270

28. Dreux M, Garaigorta U, Boyd B, Décembre E, Chung J, Whitten-Bauer C, et al. Short-range exosomal transfer of viral RNA from infected cells to plasmocytoid dendritic cells triggers innate immunity. Cells Host Microbe. 2012; 12: 558–70. doi: 10.1016/j.chom.2012.08.010

29. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter P, et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh 7.5 cells. Proc Natl Acad Sci. 2013; 110: 13109–13. doi: 10.1073/pnas.1221899110 23878230

30. Shepard D, Coudeville L, Halasa Y, Zambrano B, Dayan G. Economic impact of dengue illness in the Americas. Am J Trop Med Hyg. 2011; 84: 200–07. doi: 10.4269/ajtmh.2011.10-0503 21292885

31. Kyle J, Harris E. Global spread and persistence of dengue. Ann Rev Microbiol, 2008; 62: 71–92. doi: 10.1146/annurev.micro.62.081307.163005

32. Velandia ML, Castellanos J. Dengue virus: structure and viral cycle viral. Infectio. 2011; 15: 33–43. doi: 10.1016/S0123-9392(11)70074-1

33. Basu A, Chaturvedi U. Vascular endothelium: the battlefield of dengue viruses. FEMS Immunol Med Microbiol. 2008; 53: 287–99. doi: 10.1111/j.1574-695X.2008.00420.x 18522648

34. Srikiatkhachorn A. Plasma leakage in dengue haemorrhagic fever. Thrombo Haemost. 2009; 102: 1042–49. doi 10.1160/TH09-03-0208.

35. Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol. 1998; 161: 6338–46. doi: 10.161/11/6338 9834124

36. Pearlman E, Jiwa AH, Engleberg NC, Eisenstein BI. Growth of Legionella pneumophila in a human macrophage-like (U937) cell line. Microb Pathog. 1988; 5: 87–95. doi: 10.1016/0882-4010(88)90011-3 3237054

37. Morales L, Velandia ML, Calderón MA, Castellanos J, Chaparro-Olaya J. Polyclonal antibodies against recombinant dengue virus NS3 protein. Biomédica. 2017; 37: 131–40. doi: 10.7705/biomedica.v37i1.3249 28527257

38. Velandia-Romero ML, Acosta-Losada O, Castellanos JE. In vivo infection by a neuroinvasive neurovirulent dengue virus. J Neurovirol. 2012; 18(5): 374–87. doi: 10.1007/s13365-012-0117-y 22825914

39. Medina F, Medina J, Colón C, Vergne E, Santiago G, et al. Dengue virus: isolation, propagation, quantification, and storage. Curr Protoc Microbiol. 2012; Chapter 15: 15D.12.11–15D.12-24. doi: 10.1002/9780471729259.mc15d02s27

40. Velandia-Romero M, Calderón-Peláez MA, Castellanos J. In Vitro infection with dengue virus Induces changes in the structure and function of the mouse brain endothelium. PLoS One. 2016; 11: e0157786. doi: 10.1371/journal.pone.0157786 27336851

41. Griffiths-Jones S, Grocock R, van Dongen S, Bateman A, Enright A. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 2006; 34 (Database issue): D140–D144. doi: 10.1093/nar/gkj112 16381832

42. Chou C, Shrestha S, Yang C, Chang N, Lin Y, Liao K, et al. miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions. Nucleic Acids Res. 2017; 46(D1), D296–D302. doi: 10.1093/nar/gkx1067

43. Agarwal V, Bell G, Nam J, Bartel D. Predicting effective microRNA target sites in mammalian mRNAs. eLife. 2015; 4: e05005. doi: 10.7554/eLife.05005

44. Wang J, Lu M, Qiu C, Cui Q. TransmiR: a transcription factor-microRNA regulation database. Nucleic Acids Res. 2010; 38 (Database issue): D119–22. doi: 10.1093/nar/gkp803 19786497

45. Kutmon M1, Kelder T, Mandaviya P, Evelo CT, Coort SL. CyTargetLinker: a cytoscape app to integrate regulatory interactions in network analysis. PLoS One. 2013; 8(12): e82160. doi: 10.1371/journal.pone.0082160 24340000

46. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics. 2009; 10: 48. doi: 10.1186/1471-2105-10-48 19192299

47. Schober P, Boer C, Schwarte L. Correlation Coefficients: Appropriate use and interpretation. Anesth Analg. 2018, 126(5): 1763–1768 doi: 10.1213/ANE.0000000000002864 29481436

48. Bautista D, Rodríguez L, Franco M, Angel J, Barreto A. Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes. Cell Stress Chaperones. 2015; 20: 697–08. doi: 10.1007/s12192-015-0597-9 25975376

49. Mathivanan S, Ji H, Simpson R. Exosomes: extracellular organelles important in intercellular communication. J Proteomics. 2010; 73: 1907–20. doi: 10.1016/j.jprot.2010.06.006 20601276

50. Yang C, Tu C, Lo Y, Cheng C, Chen W. Involvement of tetraspanin C189 in cell-to-cell spreading of the dengue virus in C6/36 cells. PLoS Negl Trop Dis. 2015; 9: e0003885. doi: 10.1371/journal.pntd.0003885 26132143

51. Wu Y, Mettling C, Wu S, Yu C, Perng G, Lin Y, et al. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization. Sci Rep. 2016; 25; 6: 32243. doi: 10.1038/srep32243 27558165

52. Izquierdo-Useros N, Naranjo-Gómez M, Erkizia I, Puertas M, Borràs F, Blanco J, et al. HIV and mature dendritic cells: Trojan exosomes riding the trojan horse? Plos Pathog. 2010; 6: e1000740. doi: 10.1371/journal.ppat.1000740 20360840

53. Harding C, Geuze H. Class II molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells. J Cell Biol. 1992; 119: 531–42. doi: 10.1083/jcb.119.3.531 1400590

54. Blum J, Wearsch P, Cresswell P. Pathways of Antigen Processing. Annu Rev Immunol. 2013; 31: 443–73. doi: 10.1146/annurev-immunol-032712-095910 23298205

55. Álvarez-Rodríguez L, Ramos-Ligonio A, Rosales-Encina J, Martínez-Cázares M, Parissi-Crivelli A, López-Monteon A. Expression, purification, and evaluation of diagnostic potential and immunogenicity of a recombinant NS3 protein from all serotypes of dengue virus. J Trop Med. 2012; 2012: 956875. doi: 10.1155/2012/956875 23258983

56. Mladinich K, Piaskowski S, Rudersdorf R, Eernisse C, Weisgrau K, Martins M, et al. Dengue virus-specific CD4+ and CD8+ T lymphocytes target NS1, NS3 and NS5 in infected Indian rhesus macaques. Immunogenetics. 2012; 64: 111–21. doi: 10.1007/s00251-011-0566-0 21881953

57. Rivino L, Kumaran E, Jovanovic V, Nadua K, Teo E, Pang S, et al. Differential targeting of viral components by CD4+ versus CD8+ T lymphocytes in dengue virus infection. J Virol. 2013; 87: 2693–06. doi: 10.1128/JVI.02675-12 23255803

58. Chen Y, He L, Peng Y, Shi X, Chen J, Zhong J, et al, The hepatitis C virus protein NS3 suppresses TNF-α-stimulated activation of NF-κB by targeting LUBAC. Sci Signal. 2015; 8(403): ra118. doi: 10.1126/scisignal.aab2159 26577923

59. Zhu X, He Z, Yuan J, Wen W, Huang X, Hu Y, et al. IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection. Cell Microbiol. 2015; 17: 105–18. doi: 10.1111/cmi.12339 25131332

60. Dalvi P, Sun B, Tang N, Pulliam L. Immune activated monocyte exosomes alter microRNAs in brain endothelial cells and initiate and inflammatory response through the TLR4/MyD88 pathway. Sci Rep. 2017; 7: 9954. doi: 10.1038/s41598-017-10449-0 28855621

61. Tang N, Sun B, Gupta A, Rempel H, Pulliam L. Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-kB in endothelial cells. FASEB J. 2016; 30: 3097–06. doi: 10.1096/fj.201600368RR 27226520

62. Schmidt A, Steinritz D, Thiermann H, Meinek V, Abend M. Alteration of miRNA expression in early endothelial cells after exposure with sub-letal sulfur mustard concentrations. Toxicol Lett. 2016; 244: 88–94. doi: 10.1016/j.toxlet.2015.10.002 26456178

63. Mehta J, Mercanti F, Stone A, Wang X, Ding Z, Romeo F, et al. Gene and microRNA transcriptional signatures of angiotensin II in endothelial cells. J Cardiovasc Pharmacol. 2015; 65: 123–29. doi: 10.1097/FJC.0000000000000118 24853489

64. Ge X, Huang S, Gao H, Han Z, Chen F, Zhang S, et al. miR-21-5p alleviates leakage of injured brain microvascular endothelial barrier in vitro through suppressing inflammation and apoptosis. Brain Res. 2016; 1650: 31–40. doi: 10.1016/j.brainres.2016.07.015 27421180

65. Gu Y, Ampofo E, Menger M, Laschke M. miR-191 suppresses angiogenesis by activation of NF-kB signaling. FASEB J. 2017; 31: 3321–33. doi: 10.1096/fj.201601263R 28424351

66. Carmona A, Guerrero F, Buendia P, Obrero T, Aljama P, Carracedo J. Microvesicles derived from Indoxyl Sulphate treated endothelial cells induce endothelial progenitor cells dysfunction. Front. Physiol. 2017; 8: 666. doi: 10.3389/fphys.2017.00666 28951723

67. Ong S, Lee L, Leong Y, Ng M, Chu J. Dengue virus infection mediates HMGB1 release from monocytes involving PCAF acetylase complex and induces vascular leakage in endothelial cells. PLoS One. 2012; 7: e41932. doi: 10.1371/journal.pone.0041932 22860034

68. Bertheloot D, Latz E. HMGB1, IL-1α, IL-33 and S100 proteins: dual-function alarmins. Cell Mol Immunol. 2017; 14: 43–64; doi: 10.1038/cmi.2016.34 27569562

69. Zhu J, Wang F, Whan H, Dong N, Zhu X, Wu Y, et al. TNF-α mRNA is negatively regulated by microRNA-181a-5p in maturation of dendritic cells induced by high mobility group box-1 protein. Sci Rep. 2017; 25: 12239. doi: 10.1038/s41598-017-12492-3

70. Liu Z, Sun F, Hong Y, Liu Y, Fen M, Yin K, et al. MEG2 is regulated by miR-181a-5p and functions as a tumor suppressor gene to suppress the proliferation and migration of gastric cancer cells. Mol Cancer. 2017; 16: 133. doi: 10.1186/s12943-017-0695-7 28747184

71. Hamlin R, Rahman A, Pak T, Maringer K, Mena I, Bernal-Rubio D, et al. High-dimensional CyTOF analysis of dengue virus-infected human DCs reveals distinct viral signatures. JCI Insight. 2017; 2: pii: 92424. doi: 10.1172/jci.insight.92424 28679950

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