miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages.

Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract.

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop.

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

Acetylcholinesterase is not a generic marker of extracellular vesicles.

Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum "extra-depletion" protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.

miRNAs in platelet-poor blood plasma and purified RNA are highly stable: a confirmatory study.

OBJECTIVE: We wished to re-assess the relative stability of microRNAs (miRNAs) as compared with other RNA molecules, which has been confirmed in many contexts. When bound to Argonaute proteins, miRNAs are protected from degradation, even when released into the extracellular space in ribonucleoprotein complexes, and with or without the protection of membranes in extracellular vesicles. Purified miRNAs also appear to present less of a target for degradation than other RNAs. Although miRNAs are by no means immune to degradation, biological samples subjected to prolonged incubation at room temperature, multiple freeze/thaws, or collection in the presence of inhibitors like heparin, can typically be remediated or used directly for miRNA measurements. RESULTS: Here, we provide additional confirmation of early, well validated findings on miRNA stability and detectability. Our data also suggest that inadequate depletion of platelets from plasma may explain the occasional report that freeze-thaw cycles can adversely affect plasma miRNA levels. Overall, the repeated observation of miRNA stability is again confirmed.

Lymphocyte-Dominant Encephalitis and Meningitis in Simian Immunodeficiency Virus-Infected Macaques Receiving Antiretroviral Therapy.

A retrospective neuropathologic review of 30 SIV-infected pigtailed macaques receiving combination antiretroviral therapy (cART) was conducted. Seventeen animals with lymphocyte-dominant inflammation in the brain and/or meninges that clearly was morphologically distinct from prototypic SIV encephalitis and human immunodeficiency virus encephalitis were identified. Central nervous system (CNS) infiltrates in cART-treated macaques primarily comprised CD20+ B cells and CD3+ T cells with fewer CD68+ macrophages. Inflammation was associated with low levels of SIV RNA in the brain as shown by in situ hybridization, and generally was observed in animals with episodes of cerebrospinal fluid (CSF) viral rebound or sustained plasma and CSF viremia during treatment. Although the lymphocytic CNS inflammation in these macaques shared morphologic characteristics with uncommon immune-mediated neurologic disorders reported in treated HIV patients, including CNS immune reconstitution inflammatory syndrome and neurosymptomatic CSF escape, the high prevalence of CNS lesions in macaques suggests that persistent adaptive immune responses in the CNS also may develop in neuroasymptomatic or mildly impaired HIV patients yet remain unrecognized given the lack of access to CNS tissue for histopathologic evaluation. Continued investigation into the mechanisms and outcomes of CNS inflammation in cART-treated, SIV-infected macaques will advance our understanding of the consequences of residual CNS HIV replication in patients on cART, including the possible contribution of adaptive immune responses to HIV-associated neurocognitive disorders.

Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture.

Extracellular vesicles (EVs) are involved in intercellular communication and affect processes including immune and antiviral responses. Blood serum, a common cell culture medium component, is replete with EVs and must be depleted prior to EV-related experiments. The extent to which depletion processes deplete non-EV particles is incompletely understood, but depleted serum is associated with reduced viability and growth in cell culture. Here, we examined whether serum depleted by two methods affected HIV-1 replication. In cell lines, including HIV-1 latency models, increased HIV-1 production was observed, along with changes in cell behavior and viability. Add-back of ultracentrifuge pellets (enriched in EVs but possibly other particles) rescued baseline HIV-1 production. Primary cells were less sensitive to serum depletion processes. Virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism, surface markers, and gene expression, but not miRNA profiles, were associated with depleted serum culture. In conclusion, depleted serum conditions have a substantial effect on HIV-1 production and infectivity. Dependence of cell cultures on "whole serum" must be examined carefully along with other experimental variables, keeping in mind that the effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

Potential role of cervicovaginal extracellular particles in diagnosis of endometriosis.

BACKGROUND: Macaques are an excellent model for many human diseases, including reproductive diseases such as endometriosis. A long-recognized need for early biomarkers of endometriosis has not yet resulted in consensus. While biomarker studies have examined many bodily fluids and targets, cervicovaginal secretions have been relatively under-investigated. Extracellular vesicles (EVs, including exosomes and microvesicles) are found in every biofluid examined, carry cargo including proteins and RNA, and may participate in intercellular signaling. Little is known about EVs in the cervicovaginal compartment, including the effects of reproductive tract disease on quantity and quality of EVs. CASE PRESENTATION: In September 2014, a 9-year-old rhesus macaque was diagnosed with endometriosis at The Johns Hopkins University School of Medicine. Ultrasound-guided fine needle aspiration of a cyst and subsequent laparotomy confirmed diagnosis. The animal was sent to necropsy following euthanasia for humane reasons. Perimortem vaginal swabs and cervicovaginal lavages were obtained. Using a combination of methods, including ultracentrifugation and NanoSight visualization technology, approximate numbers of EVs from each sample were calculated and compared to populations of EVs from other, reproductively normal macaques. Fewer EVs were recovered from the endometriosis samples as compared with those from reproductively healthy individuals. CONCLUSION: To our knowledge, this is the first examination of EVs in primate cervicovaginal secretions, including those of a macaque with endometriosis. This case study suggests that additional research is justified to determine whether quantification of EVs-or their molecular cargo-in cervicovaginal lavage and vaginal swabs may provide a novel, relatively non-invasive diagnostic for primate endometrial disease or other reproductive tract diseases.

Validated MicroRNA Target Databases: An Evaluation.

Preclinical Research Positive findings from preclinical and clinical studies involving depletion or supplementation of microRNA (miRNA) engender optimism about miRNA-based therapeutics. However, off-target effects must be considered. Predicting these effects is complicated. Each miRNA may target many gene transcripts, and the rules governing imperfectly complementary miRNA: target interactions are incompletely understood. Several databases provide lists of the relatively small number of experimentally confirmed miRNA: target pairs. Although incomplete, this information might allow assessment of at least some of the off-target effects. We evaluated the performance of four databases of experimentally validated miRNA: target interactions (miRWalk 2.0, miRTarBase, miRecords, and TarBase 7.0) using a list of 50 alphabetically consecutive genes. We examined the provided citations to determine the degree to which each interaction was experimentally supported. To assess stability, we tested at the beginning and end of a five-month period. Results varied widely by database. Two of the databases changed significantly over the course of 5 months. Most reported evidence for miRNA: target interactions were indirect or otherwise weak, and relatively few interactions were supported by more than one publication. Some returned results appear to arise from simplistic text searches that offer no insight into the relationship of the search terms, may not even include the reported gene or miRNA, and may thus, be invalid. We conclude that validation databases provide important information, but not all information in all extant databases is up-to-date or accurate. Nevertheless, the more comprehensive validation databases may provide useful starting points for investigation of off-target effects of proposed small RNA therapies.

Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

BACKGROUND: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs). METHODS: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS. CONCLUSION: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.