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Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell-based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell-derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell-derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K-Akt, Ras, Hippo, TGF?, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 ?g hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue-to-airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin-challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180-196.

SUBMITTER: Tan JL

PROVIDER: S-EPMC5788876 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

Tan Jean L JL Lau Sin N SN Leaw Bryan B Nguyen Hong P T HPT Salamonsen Lois A LA Saad Mohamed I MI Chan Siow T ST Zhu Dandan D Krause Mirja M Kim Carla C Sievert William W Wallace Euan M EM Lim Rebecca R

Stem cells translational medicine 20180103 2

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell-based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell-derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell-derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibr ...[more]

PMID: 29297621

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Project description: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-1?, tumor necrosis factor-?, and prostaglandin E2 by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-??) 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-?? (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.

Project description:At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i) exosomes act as carriers of signals in utero-placental compartments and ii) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05). Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined AEC exosome characteristics and their cargo reflected the physiologic status of the cell of origin and suggests that AEC-derived exosomal p38 MAPK plays a major role in determining the fate of pregnancy. Understanding the propagation of fetal signals and their mechanisms in normal term pregnancies can provide insights into pathologic activation of such signals associated with spontaneous preterm parturitions.

Project description:Acute lung injury (ALI) induced by sepsis is characterized by disruption of the epithelial barrier and activation of alveolar macrophages (AMs), which leads to uncontrolled pulmonary inflammation. However, effective treatments for ALI are unavailable. The exact mechanism by which the initial mediator of alveolar epithelial cells (AECs) induces inflammation remains elusive. Here we investigated the roles of AEC-derived exosomes in AM activation and sepsis-induced ALI in vivo and in vitro. Cecal ligation and puncture (CLP) was utilized to establish septic lung injury model in rats. The effect of exosomal inhibition by intratracheal GW4869 administration on lung injury was investigated. To assess the effects of AEC-derived exosomes on ALI, we treated the rat alveolar epithelial cell line RLE-6TN with LPS to induce cell damage. Exosomes from conditioned medium of LPS-treated AECs (LPS-Exos) were isolated by ultracentrifugation. The miRNAs in LPS-Exos were screened by miRNA expression profile analysis. The effects of miR-92a-3p on the function of AMs were studied. We found that intratracheal GW4869 administration ameliorated lung injury following CLP-induced ALI. LPS-Exos were taken up by AMs and activated these cells. Consistently, administration of LPS-Exos in rats significantly aggravated pulmonary inflammation and alveolar permeability. Moreover, miR-92a-3p was enriched in LPS-Exos and could be delivered to AMs. Inhibition of miR-92a-3p in AECs diminished the proinflammatory effects of LPS-Exos in vivo and in vitro. Mechanistically, miR-92a-3p activates AMs along with pulmonary inflammation. This process results in activation of the NF-κB pathway and downregulation of PTEN expression, which was confirmed by a luciferase reporter assay. In conclusion, AEC-derived exosomes activate AMs and induce pulmonary inflammation mediated by miR-92a-3p in ALI. The present findings revealed a previously unidentified role of exosomal miR-92a-3p in mediating the crosstalk between injured AEC and AMs. miR-92a-3p in AEC exosomes might represent a novel diagnostic biomarker for ALI, which may lead to a new therapeutic approach.

Dataset Information

Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

Publications

Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

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