Small extracellular vesicles from the human endothelial cell line EA.hy 926 exert a self-cell activation and modulate DENV-2 genome replication and infection in naïve endothelial cells
Maria-Angelica Calderon-Pelaez, L. Johana Madroñero, Jaime E. Castellanos, Myriam L. Velandia-Romero
Abstract
Extracellular vesicles (EVs) play crucial roles in cell signaling and communication, transporting molecules that convey a message to target cells. During infectious diseases, EVs can also carry viral molecules that may contribute to viral spread, as previously reported for dengue virus (DENV). EVs from infected endothelial cells (EC) may harbor viral segments and various sets of molecules that could contribute to endothelial dysfunction during severe dengue. However, the effect of these EVs on non-infected EC (NIC) remain unknown. We characterized the EVs produced by the human EC line EA.hy 926 infected with DENV-2 and assessed their functional impact on polarized NIC.
Introduction
Cell communication strategies include soluble mediators like cytokines, chemokines [1], as well as various types of vesicles [2]. Extracellular vesicles (EVs) are spherical particles of different sizes (30–5000 nm), surrounded by a lipidic membrane. Importantly, EVs carry DNA, RNA, and small noncoding RNAs (sncRNAs) as part of their cargo, which can induce, modulate, and control a wide range of biological functions in target cells [2, 3]. It is believed that all eukaryotic cells release heterogeneous EVs to the extracellular space. Depending on their origin, EVs are called exosomes (when produced by endosomes or multivesicular bodies, MVB), or microvesicles (also known as microparticles), when produced and released from the cellular membrane [4].
Materials and method
Virus propagation in C6/36HT cells
For all the infection assays, the DENV-2 strain S3 provided by the Colombian National institute of Health (INS) was utilized [27]. The virus was propagated in C6/36HT mosquito cells (ATCC CRL-1660), which were maintained at 33°C in L15 medium (Biowest, L0300). This medium was supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (MEM NEAA, Gibco 1140–050), Tryptose phosphate broth (Gibco 18050–039), and L-Glutamine 2 mM (Biowest, X0550). Once the cells reached 80% confluence, 100 μl of viral inoculum at a multiplicity of infection (MOI) of 0.1 was diluted in fresh culture medium with 2% FBS.
Results and Discussion
EA.hy 926 cells infection with DENV-2 did not affect cell survival
Viral antigen was detected starting from 15 hpi, with a gradual increase in the number of infected cells observed over time. However, the number of infected EC was lower than observed for BHK-21 cells, which are known to be highly susceptible to DENV infection and in this case were used only as infection control. For instance, 45% of BHK cells were infected after 24h, while only 25% of EA.hy 926 cells were infected at the same time (Fig 2A). Viral replication in the EC was confirmed using the PCR technique, detecting negative viral strands in cell lysates (as viral replication signal), with a noticeable increase at 18 and 48 hpi (Fig 2B). Importantly, the infection did not affect cell survival during the evaluated times, as observed in the resazurin (Fig 2C) and LDH assays (Fig 2D). The latter showed only a 1.2% non-significant increase (p>0.05) in LDH release at 48 hpi (Fig 2D).
Acknowledgments
The authors would like to thank the Vice-chancellor of research of Universidad El Bosque for all the support and the program “Significant Experiences” for the work recognition of teachers and researchers of Universidad El Bosque, in the category of “Discovery Vocation” 2016. Also, a big thank you to Lady López and Dr. Eliana Calvo for their assessment and collaboration with the EVs proteasome assay.
Citation: Calderón-Peláez M-A, Madroñero LJ, Castellanos JE, Velandia-Romero ML (2024) Small extracellular vesicles from the human endothelial cell line EA.hy 926 exert a self-cell activation and modulate DENV-2 genome replication and infection in naïve endothelial cells. PLoS ONE 19(9): e0310735. https://doi.org/10.1371/journal.pone.0310735
Editor: Mirko Cortese, Telethon Institute of Genetics and Medicine, ITALY
Received: April 24, 2024; Accepted: September 4, 2024; Published: September 26, 2024
Copyright: © 2024 Calderón-Peláez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data supporting the findings of this study are available within the paper and its Supporting information. Additional raw data are available in the Harvard Dataverse repository: https://doi.org/10.7910/DVN/BEKZOE. Direct link: https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/BEKZOE.
Funding: MACP, JEC, MLV: Grant 113821, Award for Academic Excellence, and the Vice-chancellor of research of Universidad El Bosque, project 2018-10124. www.unbosque.edu.co The funder university did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: B50, Bortezomib 50 mM; BCA, bicinchoninic Acid; BP, biological process; Calret, calreticulin; CC, cell component; DB, dextran blue; DDX5, Probable ATP-dependent RNA helicase; DENV, dengue virus; DLS, Dynamic Light Scattering; EC, endothelial cells; EC-IC, infected endothelial cells; EC-NIC, non-infected endothelial cells; EIF2S1, eukaryotic translation initiation factor subunit 1; EVs, extracellular vesicles; FBS, fetal bovine serum; FDR, false discovery rate; Hpi, hours post-infection; Hps, hours post-seeded; IAV, influenza A virus; IC, infected endothelial cells; IFI, indirect immunofluorescence; IL-10, interleukin 10; IP-10, interferon gamma-induced protein 10; LC/MS/MS, Liquid chromatography–tandem mass spectrometry; LDH, lactate dehidrogenase; MCP-1, monocyte chemoattractant protein-1; MF, molecular functions; miRs, micro RNAs; MOI, multiplicity of infection; MVB, multivesicular bodies; MVs, microvesicles; MVs-IC, microvesicles from infected cells; MVs-NIC, microvesicles from non-infected cells; MW, molecular weight marker; N/A, not applicable; NIC, non-infected endothelial cells; NTA, Nanoparticle Tracking Analysis; PDCD6IP, Alix protein synonim; PFA, paraformaldehyde; PMSB8, proteasome subunit β type 8; RANTES, regulated on activation, normal T cell expressed and secreted; RFU, relative fluorescence units; RPM, reads per million; sEVs, small extracellular vesicles; sEVs-IC, small extracellular vesicles from infected cells; sEVs-NIC, small extracellular vesicles from non-infected cells; sncRNA, small non-coding RNA; TEER, transendothelial electric resistance; TNF-α, tumoral necrosis factor alpha; UC, ultracentrifugated; WB, western blot; WL, whole cell lysates; WL-IC, infected whole cell lysates; WL-NIC, non-infected whole cell lysates
