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.

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.

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.