Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA. aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05). While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL. These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs. This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests. N/A.

Gysler SM ，Mulla MJ ，Guerra M ，Brosens JJ ，Salmon JE ，Chamley LW ，Abrahams VM ... -  《-》

Antiphospholipid-exposed trophoblast-derived extracellular vesicles express elevated levels of TLR7/8-activating microRNAs and induce endometrial endothelial activation, in part, through TLR7.

Women with antiphospholipid syndrome (APS) are at high risk for miscarriage and preeclampsia. Unlike pro-thrombotic systemic APS, obstetric APS is associated with insufficient placentation, as well as inflammation and vascular dysfunction at the maternal-fetal interface. Antiphospholipid antibodies (aPL) can target the placental trophoblast and induce inflammation. We reported that aPL trigger trophoblast cells to produce elevated levels of IL-8 through activation of Toll-like receptor 4 (TLR4). Downstream of TLR4, we found this IL-8 response is mediated by a TLR8-activating microRNA (miR), miR-146a-3p, which is also released by the trophoblast via extracellular vesicles (EVs). Since endothelial dysfunction is a feature of obstetric APS, we sought to determine if other miRs that can activate the RNA sensors, TLR7 and/or TLR8, are released by the trophoblast via EVs after exposure to aPL, and if these EVs can activate human endometrial endothelial cells (HEECs). Using a human first trimester extravillous trophoblast cell line we found that aPL elevated their release of small EVs (<150 nm). These extracellular vesicles released from trophoblast cells exposed to aPL expressed elevated levels of TLR7/8-activating miR-21a and miR-29a, in addition to the previously reported miR-146a-3p. Extracellular vesicles from aPL-exposed human trophoblast cells triggered human endometrial endothelial cells to generate an inflammatory IL-8 response, in part through TLR7. This study highlights EVs as a mode of communication between the placenta and the maternal vasculature, as well as a potential role for TLR7/8-activating miRs in contributing to inflammation at the maternal-fetal interface in obstetric APS.

Coenen CS ，Hidalgo TN ，Lynn T ，Jones DM ，Salmon JE ，Chamley LW ，Abrahams VM ... -  《-》

Pravastatin does not prevent antiphospholipid antibody-mediated changes in human first trimester trophoblast function.

Odiari EA ，Mulla MJ ，Sfakianaki AK ，Paidas MJ ，Stanwood NL ，Gariepy A ，Brosens JJ ，Chamley LW ，Abrahams VM ... -  《-》

A role for uric acid and the Nalp3 inflammasome in antiphospholipid antibody-induced IL-1β production by human first trimester trophoblast.

Mulla MJ ，Salmon JE ，Chamley LW ，Brosens JJ ，Boeras CM ，Kavathas PB ，Abrahams VM ... -  《PLoS One》

IGF2-derived miR-483-3p contributes to macrosomia through regulating trophoblast proliferation by targeting RB1CC1.

Li J ，Fu Z ，Jiang H ，Chen L ，Wu X ，Ding H ，Xia Y ，Wang X ，Tang Q ，Wu W ... -  《-》