Amnion epithelial cell-derived exosomes induce inflammatory changes in uterine cells

Background: Fetal endocrine signals are generally considered to contribute to the timing of birth and the initiation of labor. Fetal tissues under oxidative stress release inflammatory mediators that lead to sterile inflammation within the maternal-fetal interface. Importantly, these inflammatory mediators are packaged into exosomes, bioactive cell-derived extra cellular vesicles that function as vectors and transport them from the fetal side to the uterine tissues where they deposit their cargo into target cells enhancing uterine inflammatory load. This exosome-mediated signaling is a novel mechanism for fetal-maternal communication.

Objective: This report tested the hypothesis that oxidative stress can induce fetal amnion cells to produce exosomes, which function as a paracrine intermediary between the fetus and mother and biochemically signal readiness for parturition.

Study design: Primary amnion epithelial cells were grown in normal cell culture (control) or exposed to oxidative stress conditions (induced by cigarette smoke extract). Exosomes were isolated from cell supernatant by sequential ultracentrifugation. Exosomes were quantified and characterized based on size, shape, and biochemical markers. Myometrial, decidual, and placental cells (BeWo) were treated with 2 × 10⁵, 2 × 10⁷, and 2 × 10⁹ control or oxidative stress-derived amnion epithelial cell exosomes for 24 hours. Entry of amnion epithelial cell exosomes into cells was confirmed by confocal microscopy of fluorescent-labeled exosomes. The effect of amnion epithelial cell exosomes on target cell inflammatory status was determined by measuring production of interleukin-6, interleukin-8, interleukin-1β, tumor necrosis factor-α, and prostaglandin E₂ by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-κβ) activation status by immunoblotting for phosphorylated RelA/p65. Localization of NANOG in term human myometrium and decidua obtained from women before labor and during labor was performed using immunohistochemistry. Data were analyzed by Wilcoxon-Mann-Whitney test to compare effects of exosomes from control and oxidative stress-treated amnion epithelial cells on inflammatory status of target cells.

Results: Amnion epithelial cells released ∼125 nm, cup-shaped exosomes with ∼899 and 1211 exosomes released per cell from control and oxidative stress-induced cells, respectively. Amnion epithelial cell exosomes were detected in each target cell type after treatment using confocal microscopy. Treatment with amnion epithelial cell exosomes increased secretion of interleukin-6, interleukin-8, and PGE₂ and activation of NF-κβ (each P < .05) in myometrial and decidual cells. Exosome treatments had no effect on interleukin-6 and PGE₂ production in BeWo cells. NANOG staining was higher in term labor myometrium and decidua compared to tissues not in labor.

Conclusion: In vitro, amnion epithelial cell exosomes lead to an increased inflammatory response in maternal uterine cells whereas placental cells showed refractoriness. Fetal cell exosomes may function to signal parturition by increasing maternal gestational cell inflammation.