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Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

ABSTRACT: The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000 g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000 g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000 g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

SUBMITTER: Taylor SK

PROVIDER: S-EPMC7502596 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

Taylor Sara K SK Houshdaran Sahar S Robinson Joshua F JF Gormley Matthew J MJ Kwan Elaine Y EY Kapidzic Mirhan M Schilling Birgit B Giudice Linda C LC Fisher Susan J SJ

Development (Cambridge, England) 20200908 17

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000 <b><i>g</i></b> pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to t ...[more]

PMID: 32747437

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Project description:BackgroundNano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features.ResultsDifferential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1β) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFβ. Further investigations identified CD14+ cells as major recipient cell subset of CardAP-EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells.ConclusionsCardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.

Project description:Rationale: The EGFR-driven angiogenesis is crucial in solid tumors, particularly through the delivery of biomolecules via extracellular vesicles (EVs), but the mechanism by which EGFR regulates EV cargo is still unclear. Methods: First, cell co-culture and murine tumor models were employed to examine the impact of EGFR overexpression on the pro-angiogenic properties of small EVs (sEVs) derived from oral squamous cell carcinoma (OSCC). Small RNA sequencing was then used to compare the miRNA profiles of OSCC-sEVs with and without EGFR overexpression, followed by functional enrichment and motif analyses of the differentially expressed miRNAs. Next, miRNA pull-down assays were conducted to identify potential molecules involved in sorting these miRNAs. Finally, the role of the candidate sorting protein was validated using existing public database, tissue samples, cell lines, and murine tumor models. Results: EGFR overexpression significantly enhances the pro-angiogenic effects of OSCC-sEVs, accompanied by a marked increase in the content of nucleic acid cargo carried in these sEVs. Small-RNA sequencing identified a group of miRNAs that were significantly enriched in OSCC-sEVs due to EGFR overexpression, which primarily functioned in angiogenesis and shared a characteristic "GGGU" motif. EGFR overexpression also strengthened the binding of PCBP2 with miRNAs containing this "GGGU" motif, thereby promoting their secretion through sEVs to support tumor angiogenesis. Mechanismly, EGFR overexpression upregulates PCBP2 protein content by activating its transcription rather than regulating the mRNA stability in OSCC cells. Additionally, depletion of PCBP2 impaired the EGFR-driven tumor angiogenesis by inhibiting the secretion of pro-angiogenic miRNAs through sEVs. Conclusions: EGFR boosts PCBP2 expression via transcriptional regulation, which then promotes the loading of specific miRNAs into sEVs by binding to the "GGGU" motif, thereby driving tumor angiogenesis.

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Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

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Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

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