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Page 10 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

to promote anti-inflammatory response in intestinal mucosa, probably from regulatory resident dendritic
cells. Bt OMVs can also cross mucosal barriers in the lung, suggesting that these OMVs could be used as
mucosal delivery vehicles for vaccines.

Hannah McMillan (Duke University, US) discussed bacterial vesicles in inter-kingdom communications with
plants. While relatively understudied in plants, such OMVs are known to contain virulence factors, protein
secretion components, and plant cell wall-degrading enzymes. Plant responses to bacterial pathogens are at
levels of reactive oxygen species, erection of physical barriers, and production of antimicrobial compounds.
Bacteria retaliate with the means to block and deactivate these immune responses, leading to further
immune escalation from the plant that can include systemic responses and programmed cell death. Hannah
investigated the roles of OMVs in these interactions. Using Pseudomonas syringae (Pst, plant pathogen)
and fluorescens (Pf, not pathogenic) as OMV sources, she “infected” plant leaves with OMVs. Pst OMVs
induced ICS1 protein expression (asso’ ciated with salicylate production against the bacteria). Bacterial
OMVs produced protective immunity, where plants challenged with bacteria resisted bacterial growth and
colonization, even if the non-pathogenic OMVs were used as an initial stressor. Also, both Pst OMVs and
OMVs from non-plant pathogens could protect against different pathogenic challenges. The nature of OMV-
driven protection at the level of the OMV is currently unknown and is an area of further study.

Blanca Rodriguez (Duke University, US) finished the session by talking about the immunomodulatory
impacts of RNA in Staphylococcus aureus extracellular MVs. The RNA content of MVs was originally
controversial, but has recently become generally accepted. These are mostly small RNAs; their sorting into
MVs can be affected by growth conditions and the RNAs can be transported into host cells. There are many
questions in this new area, including, the physical association of RNA with MVs, how RNAs are transmitted
to host cells, and do they modulate immunity? It is known that SA nucleic acids are immunomodulatory,
driving type I IFN responses, but the mechanism for this SA nucleic acid release and transfer is unclear,
with the hypothesis that MVs are involved. Blanca found that exogenous RNA does not bind SA MVs,
but does seem to have endogenously associated surface RNA (there is still RNA within the MVs that is
extraordinarily stable, even in the presence of proteases and nucleases). The MVs promoted IFNB expression
in macrophages; this expression was only mildly reduced if they attempted nuclease degradation prior
to incubation with macrophages. MV uptake is dynamin-dependent, as is IFNB induction, suggesting
endosomal entrapment where there are nucleic acid sensors (TLRs) that are responsible for most of the IFNB
response. Further questions include mechanisms for release of the RNA, which RNA sequences are immune
modulatory, and if alternate uptake and trafficking modes are involved.

ASEMV2020 Day 3 started with sessions on the pathology of EVs, moderated by Janusz Rak (McGill
University, Canada) and Michael Graner (CU Anschutz, US). The first speaker was Aleks Milosavljevic (Baylor
College of Medicine, US) who related the effects of glioma EVs and brain endothelial cells. Glioblastoma
(GBM), the deadliest of brain tumors, is a vascular tumor, implying tumor-driven modification of brain
endothelial cells; Aleks’ group and others asked if EV-mediated angiogenesis differed from growth factor-
driven angiogenesis. Using human brain endothelial cells as targets, EVs from GBM8 (stem cell glioma line)
enhanced vascularization patterns in the endothelial cells in ways phenotypically similar to growth factors
themselves; but which differed considerably at a molecular level involving distinct pathways. They suspected
that this was occurring at the post-transcriptome level, and therefore examined the endothelial cell fraction
that was deconvoluted from TCGA data. The results showed intersection of in vitro and in vivo perturbations.
Further analyses showed a dominant effect of growth factors over EVs in methylation/epigenetics changes.
miR-9-5p emerged from the data as a candidate extracellular RNA mediating angiogenesis upon EV exposure
to endothelial cells based on overlapping miR expression data from EVs of several sources and its plasma
biomarker status. Additionally, among downregulated gene profiles were miR-9-5p targets, including SOX7,
which could be responsible for proliferative effects. These results suggest that EV-mediated angiogenesis,