Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by antiphospholipid antibodies (aPL) together with thrombotic events and/or pregnancy morbidity (Miyakis et al., 2006, D′Cruz et al. 2007). Women with aPL are at high risk for recurrent miscarriage and pregnancy complications, such as preeclampsia; a major cause of maternal and neonatal morbidity and mortality (D′Cruz et al. 2007). Although systemic APS is a pro-thrombotic disorder, obstetric APS has a distinct pathogenesis (Viall and Chamley, 2015, Meroni et al., 2018). aPL specific for beta2 glycoprotein I (β2GPI) target the placental trophoblast leading to inflammation at the maternal-fetal interface, and placental insufficiency associated with reduced trophoblast invasion and limited uterine spiral artery remodeling (Tong et al., 2015, Viall and Chamley, 2015, Abrahams et al., 2017). However, the molecular mechanisms by which aPL impact human pregnancy remain poorly understood.

Studies from our group have illustrated the direct impact of aPL on the human trophoblast. aPL recognizing β2GPI trigger human first trimester trophoblast cells to produce elevated levels of inflammatory IL-1β and chemotactic IL-8 (Mulla et al., 2009, Mulla et al., 2013); inhibit cell migration (Mulla et al., 2010); alter angiogenic factor production (Carroll et al., 2011); and disrupt trophoblast-endothelial interactions (Alvarez et al., 2015). Thus, aPL promotes a placental pro-inflammatory, anti-migratory and anti-angiogenic profile similar to that seen in preeclampsia (Munno et al., 1999, Karumanchi and Stillman, 2006, Harris, 2010). The mechanism by which this arises are, in part, through activation of innate immune signaling, in particular for the inflammatory response. We have shown that aPL induce the trophoblast to secrete IL-1β and IL-8 via activation of Toll-like receptor (TLR) 4 (Mulla et al., 2009, Mulla et al., 2013). Downstream of TLR4 activation by aPL, inflammatory IL-1β is mediated by aPL elevating the production of endogenous uric acid, which acts as a DAMP (damage-associated molecular pattern) to subsequently activate the NLRP3 inflammasome, processing secreted IL-1β (Mulla et al., 2009, Mulla et al., 2013). In parallel and downstream of TLR4, we discovered that the increase in chemotactic IL-8 is mediated by a novel TLR8-activating microRNA (miR), miR-146a-3p, that acts as a TLR8 agonist (Gysler et al., 2016). Thus, aPL-induced miR-146a-3p and uric acid act as endogenous secondary messengers or DAMPs for trophoblast TLR8 and inflammasome activation, respectively.

In addition to miR-146a-3p, there are other miRs, with GU-rich sequences, that act non-classically by binding to and activating TLR7 and/or TLR8 (Fabbri et al., 2012); endosomal receptors typically responsible for detecting viral ssRNA (Heil et al., 2004). miR-21a, miR-29a, and Let-7b have been described as agonistic ligands for TLR7 and/or TLR8, and through these receptors can trigger inflammatory cytokine production (Fabbri et al., 2012, Lehmann et al., 2012). In this study we sought to further explore the role of TLR7/TLR8-activating miRs in aPL-driven trophoblast inflammation.

The objective of this study was to explore the role of TLR7- and TLR8-activating miRs in the aPL-driven trophoblast chemokine response, and to investigate a link between this pathway, and trophoblast inflammasome activation. For this we utilized a different trophoblast cell line and distinct molecular approaches from our original study that examined TLR8-mediated trophoblast IL-8 production in the HTR8 human first trimester trophoblast cell line (Gysler et al., 2016). Herein, we report using the Sw.71 human first trimester trophoblast cell line that aPL-induced trophoblast uric acid-triggered inflammasome activation and subsequent IL-1β secretion is mediated by miR-146a-3p activation of TLR7, and that the chemotactic IL-8 response is dependent on both TLR7 and TLR8 activation by miR-146a-3p.