Abstract
ObjectiveProtection of the fetus within the amniotic sac is primarily attained by remodeling fetal membrane (amniochorion) cells through cyclic epithelial to mesenchymal and mesenchymal to epithelial (EMT and MET) transitions. Endocrine and paracrine factors regulate EMT and MET during pregnancy. At term, increased oxidative stress forces a terminal state of EMT and inflammation, predisposing to membrane weakening and rupture. IL-6 is a constitutively expressed cytokine during gestation, but it is elevated in term and preterm births. Therefore, we tested the hypothesis that IL-6 can determine the fate of amnion membrane cells and that pathologic levels of IL-6 can cause a terminal state of EMT and inflammation, leading to adverse pregnancy outcomes.MethodsPrimary amnion epithelial cells (AECs) were treated with recombinant IL-6 (330, 1,650, 3,330, and 16,000 pg/ml) for 48 h (N = 5). IL-6-induced cell senescence (aging), cell death (apoptosis and necrosis), and cell cycle changes were studied using flow cytometry. Cellular transitions were determined by immunocytochemistry and western blot analysis, while IL-6 signaling (activation of signaling kinases) was measured by immunoassay. Inflammatory marker matrix metalloproteinase (MMP9) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations were measured using a Fluorokine E assay and ELISA, respectively. Amniotic membranes collected on gestational day (D) 12 and D18 from IL-6 knockout (KO) and control C57BL/6 mice (N = 3 each) were used to determine the impact of IL-6 on cell transitions. Fold changes were measured based on the mean of each group.ResultsIL-6 treatment of AECs at physiologic or pathologic doses increased JNK and p38MAPK activation; however, the activation of signals did not cause changes in AEC cell cycle, cellular senescence, apoptosis, necrosis, cellular transitions, or inflammation (MMP9 and GM-CSF) compared to control. EMT markers were higher on D18 compared to D12 regardless of IL-6 status in the mouse amniotic sac.ConclusionPhysiologic and pathologic concentrations of IL-6 did not cause amnion cell aging, cell death, cellular transitions, or inflammation. IL-6 may function to maintain cellular homeostasis throughout gestation in fetal membrane cells. Although IL-6 is a good biomarker for adverse pregnancies, it is not an indicator of an underlying pathological mechanism in membrane cells.
Highlights
Human fetal membranes are the innermost lining providing structure as well as immune and mechanical protection to the uterine cavity (Menon, 2016)
To test that the recombinant IL-6 used in our experiments was functionally active, primary decidua cells were treated with a range of IL-6 concentrations and the activation of the downstream transcription factor STAT3 was determined as previously shown by Devi et al (2015)
In order to evaluate the ability of IL-6s to activate various signaling pathways in amnion epithelial cells (AEC), a multiplex kinase panel was conducted
Summary
Human fetal membranes (amniochorion) are the innermost lining providing structure as well as immune and mechanical protection to the uterine cavity (Menon, 2016). A terminal state of EMT occurs at term where MET is stalled due to oxidative stress (OS)-induced senescence (Richardson et al, 2020) and accumulation of pro-EMT factors like TGFβ in membrane cells and in the amniotic fluid (Richardson et al, 2018). This leads to the accumulation of AMCs in the matrix and promotes localized inflammation and collagenolysis, leading to mechanical weakening (Richardson et al, 2020). Understanding the factors that maintain membrane cellular, structural, functional, and mechanical integrity is important
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