PLoS One. 2016 Oct 25;11(10):e0164747. doi: 10.1371/journal.pone.0164747.

Placental Chemokine Receptor D6 Is Functionally Impaired in Pre-Eclampsia.

Chiara Tersigni1, Fiorella Di Nicuolo1, Giuseppe Maulucci2, Alessandro Rolfo3, Domenica Giuffrida3, Manuela Veglia1, Marco De Spirito2, Giovanni Scambia1, Tullia Todros3, Nicoletta Di Simone1.

1. Department of Obstetrics and Gynaecology, Fondazione Policlinico Agostino Gemelli, Università Cattolica Del Sacro Cuore, Rome, Italy.
2. Institute of Physics, Università Cattolica Del Sacro Cuore, Rome, Italy.
3. Department of Surgical Sciences, Sant’Anna Hospital, Università degli Studi di Torino, Turin, Italy.

Correspondence should be addressed to Nicoletta Di Simone, Department of Obstetrics and Gynaecology, Fondazione Policlinico Agostino Gemelli, Università Cattolica Del Sacro Cuore, 00168 Rome, Italy. E.mail:



D6 is a chemokines scavenger receptor that binds with high affinity CC chemokines, internalizes and targets the ligands for degradation. It is expressed in trophoblast-derived tissues and prevents excessive placenta leukocyte infiltration. The aim of this study was to investigate the expression and function of D6 in human placentae from pre-eclampsia (PE) and healthy pregnant women.

Plasma levels of D6-binding CC chemokines (CCL-2, CCL-3, CCL-4, CCL-7, CCL-11) and pro-inflammatory cytokines (IL-6, TNF-α, CRP) were analyzed in 37 healthy pregnant women and 38 patients with PE by multiplex bead assay. Higher circulating levels of CCL7, CCL11, IL-6, (p<0.0001) and CRP (p<0.05) were observed in PE women compared to controls. Levels of circulating CCL4 were decreased in PE (p<0.001), while no significant differences of CCL2, CCL3 or TNF-α levels were detected. Immunofluorescent staining of placental sections showed higher expression of D6 receptor in the PE syncytiotrophoblast. Confocal and Western blot (WB) analyses revealed a membrane distribution of D6 in trophoblast cells in PE. Increased activation of D6 intracellular pathway was observed by Western blot analyses of p-LIMK and p-cofilin in trophoblast cell lysates. D6 functional assays showed reduced scavenging of CCL2 in PE cells compared to controls. Since actin filaments spatial assembling is essential for D6 intracellular trafficking and scavenging activity, we investigated trophoblast cytoskeleton organization by confocal microscopy and we observed a dramatic disarrangement in PE compared to controls.

Our results suggest membrane distribution of D6 receptor on trophoblast cell membranes in PE, together with reduced functionality, probably due to trophoblast cells cytoskeleton impairment.




Pre-eclampsia (PE) is a pregnancy-specific hypertensive disorder defined as new onset hypertension and proteinuria at or after 20 weeks’ gestation [1]. Complicating 2-8% of all pregnancies, PE is a major cause of maternal morbidity and mortality and of adverse perinatal outcomes [2]. The underlying causes remain unclear but it has been recognized to be a placenta-driven disorder associated with poor placental perfusion causing hypoxia-reperfusion injury and oxidative syncytiotrophoblast stress. Release into the maternal circulation of placental pro-inflammatory and anti-angiogenic factors ensues, leading to endothelial dysfunction, exaggerated maternal inflammatory response and hypercoagulability [3-5]. The systemic inflammatory response occurring in overt PE involves leukocytes, the clotting and complement systems and the endothelium. Communication between these various components of the inflammatory network is facilitated by a large variety of secreted proteins such as cytokines. Among these, chemokines are essential for leukocyte chemoattraction [6,7]. D6 decoy receptor is a chemokine receptor that binds most inflammatory, but not homeostatic, CC chemokines, internalizes constitutively, and targets the ligand for degradation [8,9]. In resting conditions, D6 is predominantly located in intracellular/perinuclear compartments and only 5% is detectable on the cell surface [10,11]. After chemokines binding, D6 is constitutively internalized and then targeted to early endosomes [8,12]. Once D6 has been internalized, ligands dissociate from the receptor and are targeted to degradation in lysosomal compartments, while the receptor is free to recycle back to the cell surface [11-13] with mechanisms that are strictly dependent on cytoskeleton dynamics [14]. Indeed, the engagement of D6 receptor by its ligands activates a β-arrestin1-dependent G protein-independent signaling pathway, the Rac1-p21-activated kinase 1 (PAK1)-LIM kinase 1(LIMK1) cascade [14]. This cascade results in the phosphorylation and inactivation of a major actin-depolymerizing factor, cofilin, that enable actin network rearrangements that are critically required for the increased abundance of D6 protein on the cell surface and for its chemokine-scavenging activity [14].

Differently from other chemokine receptors, D6 expression has been reported mainly in non-hematopoietic cells and includes endothelial cells lining afferent lymphatic in skin, gut, and lung [15]. D6 expression has been also detected in the human placenta [16], particularly concentrated toward the apical surface of chorionic villous trophoblast, the side directly contacting maternal blood [17]. Recently, a role for D6 decoy receptor activity in the maintenance of controlled inflammatory placental environment at maternal-fetal interface has been proposed by Martinez de la Torre and co-authors, who showed that trophoblast cells express D6 and use this molecule to scavenge inflammatory CC chemokines [18]. Intriguingly, they also provided evidences that D6 is required to prevent excessive placenta leukocyte infiltration and inflammation- and autoantibody-induced fetal loss in animal models, thus protecting the fetus from miscarriage [18]. Consistently, Madigan et al. have demonstrated that, in normal pregnancy, despite robust expression of pro-inflammatory chemokines by gestational tissues, D6-binding chemokines are less abundant in maternal plasma compared to non-pregnant women. Indeed, maternal blood continuously flows towards D6-expressing chorionic villi, suggesting a crucial role for D6 decoy receptor in blood chemokines scavenging and regulation of local and systemic inflammation [17]. Conversely, a trend of increase in circulating D6-binding chemokines was observed in the third trimester in 34 women later developing PE compared to gestational age-matched controls [17]. Since PE is a placental-induced inflammatory disorder, characterized by higher circulating blood levels of pro-inflammatory cytokines (CCL2, IL-6, IL-8, TNF-α, RANTES and MIF) [17, 19-23], the aim of this study was to investigate a possible abnormal expression or function of placental D6 receptor, in human placentae obtained from women with PE compared to women with normal pregnancies. With this purpose, we collected blood and placentae from 37 women with overt PE and 38 healthy pregnant women just before delivery and we quantified circulating pro-inflammatory cytokines (IL-6,TNF-α, CRP) and chemokines, specifically scavenged by D6 receptor (CCL-2, CCL-3, CCL-4, CCL-7, CCL-11). Placental lysates and placental sections were analyzed for D6 expression by Western blot analysis and immunofluorescent staining, respectively. Primary trophoblast cells obtained from PE women and controls were evaluated for D6 cellular distribution by confocal microscopy and cell membranes Western blot analysis. Furthermore, we investigated the functional activity of D6 in trophoblast cells by both CCL-2 (D6-binding chemokine) binding and scavenging assays. Finally, we analyzed by confocal microscopy trophoblast actin fibers organization, whose integrity is crucial for D6 internalization and scavenging. In the complex, our results indicates that D6 is concentrated on trophoblast cell membranes in PE, consistently with higher circulating levels of D6-ligand chemokines, but its scavenging activity is reduced due to trophoblast cytoskeleton disarrangement and, thus, impairment of receptor internalization and recycling dynamics.



Figure 1. Representative image showing D6 expression in primary trophoblast cell cultures from normal pregnant compared to PE women. Confocal microscopy analysis of cells from PE women (B) showed a more intense expression of D6 (green) on cell membranes, together with a more diffuse cytoplasm distribution compared to control cells (A), that displayed atypical speckled cytoplasmic D6 positivity. Scale bar 20 µm. Quantification of D6 expression on trophoblast cell membranes was performed by (C) confocal analysis of fluorescence on membranes, as percentage of the whole cells fluorescence, and (D) Western blot analysis of D6 on cell membrane lysates. Expression of both 46 and 49 KDa isoforms of D6 was increased in trophoblast membranes in PE. Results are expressed as mean ± SE of six experiments.*p<0.05; ***p<0.001.



Figure 2. Representative image of cytoskeleton F-actin fibers organization in primary trophoblast cell cultures from placentae of healthy pregnant women at term (A) or PE women (B). Confocal analysis of trophoblast cells cytoskeleton organization, performed by intracellular F-actin FITC-staining, displayed a dramatic disarrangement and loss of spatial orientation of actin fibers in PE trophoblast cells but not in control cells. Scale bar 20 µm.



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