SUMMARYExtracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication, being involved in a wide array of key biological processes. Eukaryotic cells, and also bacteria, actively release heterogeneous subtypes of EVs into the extracellular space, where their contents reflect their (sub)cellular origin and the physiologic state of the parent cell. Within the past 20 years, presumed subtypes of EVs have been given a rather confusing diversity of names, including exosomes, microvesicles, ectosomes, microparticles, virosomes, virus-like particles, and oncosomes, and these names are variously defined by biogenesis, physical characteristics, or function. The latter category, functions, in particular the transmission of biological signals between cellsin vivoand how EVs control biological processes, has garnered much interest. EVs have pathophysiological properties in cancer, neurodegenerative disorders, infectious disease, and cardiovascular disease, highlighting possibilities not only for minimally invasive diagnostic applications but also for therapeutic interventions, like macromolecular drug delivery. Yet, in order to pursue therapies involving EVs and delivering their cargo, a better grasp of EV targeting is needed. Here, we review recent progress in understanding the molecular mechanisms underpinning EV uptake by receptor-ligand interactions with recipient cells, highlighting once again the overlap of EVs and viruses. Despite their highly heterogeneous nature, EVs require common viral entry pathways, and an unanticipated specificity for cargo delivery is being revealed. We discuss the challenges ahead in delineating specific roles for EV-associated ligands and cellular receptors.

Background and objectivesThe cause of systemic lupus erythematosus (SLE), a severe autoimmune disease, remains unclear. Genetic predisposition cannot be the only explanation for the emergence of SLE and environmental factors could contribute to the complex aetiology of this disease. Epstein-Barr virus (EBV) infection has been implied as a possible factor in early SLE pathogenesis. This study aimed to obtain anti-EBV antibody characteristics in a longitudinal SLE serum cohort, to find correlations between anti-EBV reactivity and disease activity, including lupus nephritis (LN), and to show their clinical utility as a diagnostic marker in SLE.Materials and methodsBlood samples were taken yearly from patients included in the Amsterdam SLE cohort (start 2007). Longitudinal characteristics of the anti-EBV antibody profile in SLE patient serum (n = 30), were tested blind with the immunoblot technique, using 3 mm nitrocellulose strips produced with SDS page and electrophoretic transfer of EBV lytic phase antigens. EBV antigens were obtained from nuclear extract of HH514.c16 cells induced with TPA/sodium butyrate for expression of EBV lytic phase. Patterns were compared to clinical characteristics, including SLEDAI (SLE disease activity index) and occurrence of lupus nephritis.ResultsWe found that immune-reactivity against EBV antigens in the serum of SLE is highly perturbed when compared to healthy individuals. Virtually all patients had positive reactivity of IgG antibodies against latent (EBNA1) and lytic (EAd, Zebra, VCA) EBV associated antigens. The patterns of reactivity changed overtime and are possibly related to disease activity (SLEDAI). Strikingly, SLE patients that develop lupus nephritis seem to have an altered serum reactivity for currently unknown antigens, up to two years before clinical diagnosis.ConclusionsOur results strongly suggest associations of EBV antibody reactivity in SLE patients. This suggests aberrant EBV control in all SLE patients but particular in those with renal involvement. The results warrant larger studies measuring EBV serum reactivity as a possible prognostic biomarker for renal involvement in SLE and response to treatment.

BackgroundThe pathogenesis of Systemic Lupus Erythematosus (SLE) is incompletely understood. Low concordance rates in monozygotic twins and geographical differences in disease risk, suggest involvement of environmental factors in the etiology of SLE. Virtually all patients with SLE have increased antibody titers against EBV antigens, chronically amplified infection frequencies, and 99-100% of young SLE patients seroconvert against Epstein-Barr virus, compared to only 70% of controls. However, a specified role for EBV in SLE pathogenesis remains unclear. Recently, we discovered that EBV-infected B cells secrete highly inflammatory viral RNAs (EBERs) via extracellular vesicles (EVs).ObjectivesWe aimed to investigate the extent of anti-EBV AB responses in SLE patients and study the potential pro-inflammatory role of EBV-secreted, EBER1-laden extracellular vesicles and assess EBV-RNAs as biomarkers in SLE tissues.MethodsWe explored the molecular basis of altered anti-EBV antibody responses at the epitope level, by immunoblot and peptide-based ELISA in serial samples of SLE patients. We determined EBERs with RT-PCR and in situ hybridization in blood, urine and affected tissue (renal, skin) of SLE patients (with or without nephritis). The inflammatory properties of EBV-EVs were studied in vitro using primary cultures.ResultsWe demonstrate that aberrant antibodies against EBV-antigens and presence of extracellular EBV small RNA (EBER1) distinguish SLE patient sera from rheumatoid arthritis patients and healthy individuals. SLE patients with renal involvement have a distinct pattern of anti-EBV antibody reactivity and lack detectable EBER1 in circulating plasma exosomes. However, surprisingly high levels of EBER1 were detected in lupus nephritis (LN) biopsy material, but not in IgA nephritis tissue. This is compatible with an exogenous (blood-born) source of EBER1, because EBV DNA was near absent and similar observations were made in SLE-associated skin lesions. In situ hybridization showed EBER1 positivity in tubular epithelial cells (TECs). Purified EVs from latent EBV-infected cells are internalized by both primary TEC and plasmacytoid dendritic cells in a phosphatidylserine receptor-dependent manner, causing pro-inflammatory IL-6 production upon EBER1 recognition by viral sensors.ConclusionsEBV-infection is deregulated in SLE patients based on anti-EBV Ab responses and elevated levels of extracellular EBER1 in circulation that targets the renal epithelium and skin. Our findings in vitro confirm strong pro-inflammatory functions of EBER1-containing exosomes that target specific cell types, shedding new light on EBV as co-factor in the pathophysiology of SLE and the role of EVs in autoimmune disease.Disclosure of InterestNone declared